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Redistribution of the water channel protein aquaporin-4 and the K + channel protein Kir4.1 differs in low- and high-grade human brain tumors
Arne Warth; Michel Mittelbronn; Hartwig Wolburg
The blood-brain barrier (BBB) regulation is characterized by an interplay between endothelial cells, subendothelial basal laminae and astrocytic cells. Astroglial cells are highly polarized by the differentiation of perivascular membrane domains. These domains are characterized by the aggregation of, among other molecules, the water channel protein aquaporin-4 (AQP4), the dystrophin-dystroglycan complex, and the inwardly rectifying potassium channel protein Kir4.1. Normally,
Water channel proteins AQP3 and AQP9 are present in syncytiotrophoblast of human term placenta.
Damiano, A; Zotta, E; Goldstein, J; Reisin, I; Ibarra, C
The syncytiotrophoblast of human term placenta (HST) is a continuous, multinucleated structure with minimal tight junctions, which results from the fusion of the underlying cytotrophoblast cells. Consequently, the transport of metabolites, ions and water from mother to fetus could take place primarily via transcellular routes. Transcellular water flux may be facilitated by aquaporins, membrane proteins functioning as water channels that are widely expressed in cells and tissues. Here, we report the presence of AQP3 and AQP9 in the apical membranes of HST using RT-PCR, immunoblotting and immunohistochemistry. Since AQP3 is not only a water channels, but also permits the rapid passage of both urea and glycerol, while AQP9 also mediates the passage of carbamides, polyols, purines, and pyrimidines, we have speculated that these proteins could be involved in the transport of water and solutes from mother to fetus. PMID:11597198
Molecular Cloning, Overexpression and Characterization of a Novel Water Channel Protein from Rhodobacter sphaeroides
Erbakan, Mustafa; Shen, Yue-xiao; Grzelakowski, Mariusz; Butler, Peter J.; Kumar, Manish; Curtis, Wayne R.
Aquaporins are highly selective water channel proteins integrated into plasma membranes of single cell organisms; plant roots and stromae; eye lenses, renal and red blood cells in vertebrates. To date, only a few microbial aquaporins have been characterized and their physiological importance is not well understood. Here we report on the cloning, expression and characterization of a novel aquaporin, RsAqpZ, from a purple photosynthetic bacterium, Rhodobacter sphaeroides ATCC 17023. The protein was expressed homologously at a high yield (?20 mg/L culture) under anaerobic photoheterotrophic growth conditions. Stopped-flow light scattering experiments demonstrated its high water permeability (0.17▒0.05 cm/s) and low energy of activation for water transport (2.93▒0.60 kcal/mol) in reconstituted proteoliposomes at a protein to lipid ratio (w/w) of 0.04. We developed a fluorescence correlation spectroscopy based technique and utilized a fluorescent protein fusion of RsAqpZ, to estimate the single channel water permeability of RsAqpZ as 1.24 (▒0.41) x 10?12 cm3/s or 4.17 (▒1.38)ū1010 H2O molecules/s, which is among the highest single channel permeability reported for aquaporins. Towards application to water purification technologies, we also demonstrated functional incorporation of RsAqpZ in amphiphilic block copolymer membranes. PMID:24497982
On the definition, nomenclature and classification of water channel proteins (aquaporins and relatives).
Benga, Gheorghe
A water channel protein (WCP) or a water channel can be defined as a transmembrane protein that has a specific three-dimensional structure with a pore that provides a pathway for water permeation across biological membranes. The pore is formed by two highly conserved regions in the amino acid sequence, called NPA boxes (or motifs) with three amino acid residues (asparagine-proline-alanine, NPA) and several surrounding amino acids. The NPA boxes have been called the "signature" sequence of WCPs. WCPs are a family of proteins belonging to the Membrane Intrinsic Proteins (MIPs) superfamily. In addition, in the MIP superfamily (with more than 1000 members) there are also proteins with no channel activity. The WCP family include three subfamilies: aquaporins, aquaglyceroporins and S-aquaporins. (1) The aquaporins (AQPs) are water selective or specific water channels, also named by various authors as "orthodox", "ordinary", "conventional", "classical", "pure", "normal", or "sensu strictu" aquaporins); (2) The aquaglyceroporins are permeable to water, but also to other small uncharged molecules, in particular glycerol; this family includes the glycerol facilitators, abbreviated as GlpFs, from glycerol permease facilitators. The "signature" sequence for aquaglyceroporins is the aspartic acid residue (D) in the second NPA box. (3) The third subfamily of WCPs have little conserved amino acid sequences around the NPA boxes, unclassifiable to the first two subfamilies. I recommend to use always for this subfamily the name S-aquaporins. They are also named "superaquaporins", "aquaporins with unusual (or deviated) NPA boxes", "subcellular aquaporins", or "sip-like aquaporins". I also recommend to use always the spelling aquaporin (not aquaporine), and, for various AQPs, the abbreviation AQP followed immediately by the number, (e.g. AQP1), with no space or--which might create confusions with "minus". PMID:22542572
Developmental Gene Expression and Tissue Distribution of the CHIP28 Water Channel Protein
Carolyn Bondy; Edward Chin; Barbara L. Smith; Gregory M. Preston; Peter Agre
The CHIP28 water channel is a major component of red cell and renal tubule membranes; however, its ontogeny and tissue distribution remain undefined. Three patterns of expression were identified when CHIP28 mRNA was surveyed by in situ hybridization histochemistry in rats between embryonic day 14 and maturity. (i) CHIP28 mRNA and protein were very abundant in hematopoietic tissue and kidneys
Molecular and functional characterization of multiple aquaporin water channel proteins from the western tarnished plant bug, Lygus hesperus
Technology Transfer Automated Retrieval System (TEKTRAN)
Aquaporins (AQPs) are integral membrane channel proteins that facilitate the bidirectional transfer of water or other small solutes across biological membranes involved in numerous essential physiological processes. In arthropods, AQPs belong to several subfamilies, which contribute to osmoregulatio...
The Role of Water Channel Proteins in Facilitating Recovery of Leaf Hydraulic Conductance from Water Stress in Populus trichocarpa
Laur, Joan; Hacke, Uwe G.
Gas exchange is constrained by the whole-plant hydraulic conductance (Kplant). Leaves account for an important fraction of Kplant and may therefore represent a major determinant of plant productivity. Leaf hydraulic conductance (Kleaf) decreases with increasing water stress, which is due to xylem embolism in leaf veins and/or the properties of the extra-xylary pathway. Water flow through living tissues is facilitated and regulated by water channel proteins called aquaporins (AQPs). Here we assessed changes in the hydraulic conductance of Populus trichocarpa leaves during a dehydration-rewatering episode. While leaves were highly sensitive to drought, Kleaf recovered only 2 hours after plants were rewatered. Recovery of Kleaf was absent when excised leaves were bench-dried and subsequently xylem-perfused with a solution containing AQP inhibitors. We examined the expression patterns of 12 highly expressed AQP genes during a dehydration-rehydration episode to identify isoforms that may be involved in leaf hydraulic adjustments. Among the AQPs tested, several genes encoding tonoplast intrinsic proteins (TIPs) showed large increases in expression in rehydrated leaves, suggesting that TIPs contribute to reversing drought-induced reductions in Kleaf. TIPs were localized in xylem parenchyma, consistent with a role in facilitating water exchange between xylem vessels and adjacent living cells. Dye uptake experiments suggested that reversible embolism formation in minor leaf veins contributed to the observed changes in Kleaf. PMID:25406088
ERIC Educational Resources Information Center
White, A. S.
Describes a simple water channel, for use with an overhead projector. It is run from a water tap and may be used for flow visualization experiments, including the effect of streamlining and elementary building aerodynamics. (MLH)
Identification and characterization of functional aquaporin water channel protein from alimentary tract of whitefly, Bemisia tabaci
Technology Transfer Automated Retrieval System (TEKTRAN)
Some hemipteran xylem and phloem feeding insects have evolved specialized alimentary structures or filter chambers that rapidly transport water for excretion or osmoregulation. In the whitefly, Bemisia tabaci, mass movement of water through opposing alimentary tract tissues within the filter chamber...
Conversion of a mechanosensitive channel protein from a membrane-embedded to a water-soluble form by covalent modification with amphiphiles.
Becker, Christian F W; Strop, Pavel; Bass, Randal B; Hansen, Kirk C; Locher, Kaspar P; Ren, Gang; Yeager, Mark; Rees, Douglas C; Kochendoerfer, Gerd G
Covalent modification of integral membrane proteins with amphiphiles may provide a general approach to the conversion of membrane proteins into water-soluble forms for biophysical and high-resolution structural studies. To test this approach, we mutated four surface residues of the pentameric Mycobacterium tuberculosis mechanosensitive channel of large conductance (MscL) to cysteine residues as anchors for amphiphile attachment. A series of modified ion channels with four amphiphile groups attached per channel subunit was prepared. One construct showed the highest water solubility to a concentration of up to 4mg/ml in the absence of detergent. This analog also formed native-like, alpha-helical homo-pentamers in the absence of detergent as judged by circular dichroism spectroscopy, size-exclusion chromatography and various light-scattering techniques. Proteins with longer, or shorter polymers attached, or proteins modified exclusively with polar cysteine-reactive small molecules, exhibited reduced to no solubility and higher-order aggregation. Electron microscopy revealed a homogeneous population of particles consistent with a pentameric channel. Solubilization of membrane proteins by covalent attachment of amphiphiles results in homogeneous particles that may prove useful for crystallization, solution NMR spectroscopy, and electron microscopy. PMID:15465059
Channel incision and water quality
NASA Astrophysics Data System (ADS)
Shields, F. D.
Watershed development often triggers channel incision that leads to radical changes in channel morphology. Although morphologic evolution due to channel incision has been documented and modeled by others, ecological effects, particularly water quality effects, are less well understood. Furthermore, environmental regulatory frameworks for streams frequently focus on stream water quality and underemphasize hydrologic and geomorphic issues. Discharge, basic physical parameters, solids, nutrients (nitrogen and phosphorus), chlorophyll and bacteria were monitored for five years at two sites along a stream in a mixed cover watershed characterized by rapid incision of the entire channel network. Concurrent data were collected from two sites on a nearby stream draining a watershed of similar size and cultivation intensity, but without widespread incision. Data sets describing physical aquatic habitat and fish fauna of each stream were available from other studies. The second stream was impacted by watershed urbanization, but was not incised, so normal channel-floodplain interaction maintained a buffer zone of floodplain wetlands between the study reach and the urban development upstream. The incised stream had mean channel depth and width that were 1.8 and 3.5 times as large as for the nonincised stream, and was characterized by flashier hydrology. The median rise rate for the incised stream was 6.4 times as great as for the nonincised stream. Correlation analyses showed that hydrologic perturbations were associated with water quality degradation, and the incised stream had levels of turbidity and solids that were two to three times higher than the nonincised, urbanizing stream. Total phosphorus, total Kjeldahl N, and chlorophyll a concentrations were significantly higher in the incised stream, while nitrate was significantly greater in the nonincised, urbanizing stream (p < 0.02). Physical aquatic habitat and fish populations in the nonincised urbanizing stream were superior, as it supported almost twice as many species and yielded more than four times as much biomass per unit of effort. These results suggest that channel incision is associated with a complex of ecological stressors that includes channel erosion, hydrologic perturbation, and water quality and physical habitat degradation. Ecological engineering of stream corridors must focus at least as much energy on mediating hydrologic perturbations and managing habitat quality as on pollutant loadings.
Regulation of Ion Channels by G Proteins
NSDL National Science Digital Library
Maria Diverse-Pierluissi (Mount Sinai School of Medicine.; Department of Pharmacology and Biological Chemistry REV)
This Teaching Resource provides lecture notes and slides for a class covering regulation of ion channels by G proteins and is part of the course "Cell Signaling Systems: A Course for Graduate Students." The lecture begins with an overview of calcium channels and then proceeds to describe the interaction of signaling molecules with calcium channels.
Membrane-Protein Interactions in Mechanosensitive Channels
Paul Wiggins; Rob Phillipsy
In this article, we examine the mechanical role of the lipid bilayer in ion channel conformation and function with specific reference to the case of the mechanosensitive channel of large conductance (MscL). In a recent article we argued that mechanotransduction very naturally arises from lipid-protein interactions by invoking a simple analytic model of the MscL channel and the surrounding lipid
A substrate channel in the nitrogenase MoFe protein
Brett M. Barney; Michael G. Yurth; Patricia C. Dos Santos; Dennis R. Dean; Lance C. Seefeldt
Nitrogenase catalyzes the six electron\\/six proton reduction of N2 to two ammonia molecules at a complex organometallocluster called ōFeMo cofactor.ö This cofactor is buried within the ?-subunit\\u000a of the MoFe protein, with no obvious access for substrates. Examination of high-resolution X-ray crystal structures of MoFe\\u000a proteins from several organisms has revealed the existence of a water-filled channel that extends from
Phylogenetic Characterization of the MIP Family of Transmembrane Channel Proteins
J. H. Park
.ĀĀ The ubiquitous major intrinsic protein (MIP) family includes several transmembrane channel proteins known to exhibit specificity\\u000a for water and\\/or neutral solutes. We have identified 84 fully or partially sequenced members of this family, have multiply\\u000a aligned over 50 representative, divergent, fully sequenced members, have used the resultant multiple alignment to derive current\\u000a MIP family-specific signature sequences, and have constructed
Aquaporin water channels in gastrointestinal physiology
Ma, Tonghui; Verkman, A S
Fluid transport is a major function of the gastrointestinal (GI) tract with more than 9 litres of fluid being absorbed or secreted across epithelia in human salivary gland, stomach, the hepatobiliary tract, pancreas, small intestine and colon. This review evaluates the evidence that aquaporin-type water channels are involved in GI fluid transport. The aquaporins are a family of small (?30 kDa) integral membrane proteins that function as water channels. At least seven aquaporins are expressed in various tissues in the GI tract: AQP1 in intrahepatic cholangiocytes, AQP4 in gastric parietal cells, AQP3 and AQP4 in colonic surface epithelium, AQP5 in salivary gland, AQP7 in small intestine, AQP8 in liver, pancreas and colon, and AQP9 in liver. There are functional data suggesting that some GI cell types expressing aquaporins have high or regulated water permeability; however, there has been no direct evidence for a role of aquaporins in GI physiology. Recently, transgenic mice have been generated with selective deletions of various aquaporins. Preliminary evaluation of GI function suggests a role for AQP1 in dietary fat processing and AQP4 in colonic fluid absorption. Further study of aquaporin function in the GI tract should provide new insights into normal GI physiology and disease mechanisms, and may yield novel therapies to regulate fluid movement in GI diseases. PMID:10332084
Aquaporin1, Nothing but a Water Channel
Satoshi P. Tsunoda; Burkhard Wiesner; Dorothea Lorenz; Walter Rosenthal; Peter Pohl; Freie Universitaet
Aquaporin-1 (AQP1) is a membrane channel that al- lows rapid water movement driven by a transmembrane osmotic gradient. It was claimed to have a secondary function as a cyclic nucleotide-gated ion channel. How- ever, upon reconstitution into planar bilayers, the ion channel exhibited a 10-fold lower single channel con- ductance than in Xenopus oocytes and a 100-fold lower open probability
Glucose Transporters Serve as Water Channels
Jorge Fischbarg; Kunyan Kuang; Juan C. Vera; Suzanne Arant; Samuel C. Silverstein; John Loike; Ora M. Rosen
Water traverses the plasma membranes of some eukaryotic cells faster than can be explained by the water permeability of their lipid bilayers. This has led to a search for a water channel. Our previous work identified glucose transporters as candidates for such a channel. We report here that Xenopus laevis oocytes injected with mRNA encoding the brain\\/Hep G2, adult skeletal
Molecular dynamics simulations of water within models of ion channels.
Breed, J; Sankararamakrishnan, R; Kerr, I D; Sansom, M S
The transbilayer pores formed by ion channel proteins contain extended columns of water molecules. The dynamic properties of such waters have been suggested to differ from those of water in its bulk state. Molecular dynamics simulations of ion channel models solvated within and at the mouths of their pores are used to investigate the dynamics and structure of intra-pore water. Three classes of channel model are investigated: a) parallel bundles of hydrophobic (Ala20) alpha-helices; b) eight-stranded hydrophobic (Ala10) antiparallel beta-barrels; and c) parallel bundles of amphipathic alpha-helices (namely, delta-toxin, alamethicin, and nicotinic acetylcholine receptor M2 helix). The self-diffusion coefficients of water molecules within the pores are reduced significantly relative to bulk water in all of the models. Water rotational reorientation rates are also reduced within the pores, particularly in those pores formed by alpha-helix bundles. In the narrowest pore (that of the Ala20 pentameric helix bundle) self-diffusion coefficients and reorientation rates of intra-pore waters are reduced by approximately an order of magnitude relative to bulk solvent. In Ala20 helix bundles the water dipoles orient antiparallel to the helix dipoles. Such dipole/dipole interaction between water and pore may explain how water-filled ion channels may be formed by hydrophobic helices. In the bundles of amphipathic helices the orientation of water dipoles is modulated by the presence of charged side chains. No preferential orientation of water dipoles relative to the pore axis is observed in the hydrophobic beta-barrel models. Images FIGURE 1 FIGURE 5 FIGURE 7 PMID:8785323
Origin of Martian channels - Clathrates and water
NASA Technical Reports Server (NTRS)
Peale, S. J.; Schubert, G.; Lingenfelter, R. E.
Criticism is directed at the suggestion that Martian channels may have been eroded by liquid water produced by the depressurization of CO2 hydrate. The release of pre-existing subsurface liquid water, such as that trapped under a permafrost layer, by meteorite impact or tectonic activity could produce sufficient flow and would not require heat transfer. The presence of water in a CO2 hydrate is shown to be detrimental to its release from an underground reservoir.
The AQP2 water channel: Effect of vasopressin treatment, microtubule disruption, and distribution in neonatal rats
I. Saboli?; T. Katsura; J.-M. Verbavatz; D. Brown
Aquaporin 2 is a collecting duct water channel that is located in apical vesicles and in the apical plasma membrane of collecting duct principal cells. It shares 42% identity with the proximal tubule\\/thin descending limb water channel, CHIP28. The present study was aimed at addressing three questions concerning the location and behavior of the AQP2 protein under different conditions. First,
Water at interface with proteins
Giancarlo Franzese; Valentino Bianco; Svilen Iskrov
Water is essential for the activity of proteins. However, the effect of the properties of water on the behavior of proteins is only partially understood. Recently, several experiments have investigated the relation between the dynamics of the hydration water and the dynamics of protein. These works have generated a large amount of data whose interpretation is debated. New experiments measure the dynamics of water at low temperature on the surface of proteins, finding a qualitative change (crossover) that might be related to the slowing down and stop of the protein's activity (protein glass transition), possibly relevant for the safe preservation of organic material at low temperature. To better understand the experimental data several scenarios have been discussed. Here, we review these experiments and discuss their interpretations in relation with the anomalous properties of water. We summarize the results for the thermodynamics and dynamics of supercooled water at an interface. We consider also the effect of water on protein stability, making a step in the direction of understanding, by means of Monte Carlo simulations and theoretical calculations, how the interplay of water cooperativity and hydrogen bonds interfacial strengthening affects the protein cold denaturation.
Protein-water displacement distributions.
Doster, Wolfgang; Settles, Marcus
The statistical properties of fast protein-water motions are analyzed by dynamic neutron scattering experiments. Using isotopic exchange, one probes either protein or water hydrogen displacements. A moment analysis of the scattering function in the time domain yields model-independent information such as time-resolved mean square displacements and the Gauss-deviation. From the moments, one can reconstruct the displacement distribution. Hydration water displays two dynamical components, related to librational motions and anomalous diffusion along the protein surface. Rotational transitions of side chains, in particular of methyl groups, persist in the dehydrated and in the solvent-vitrified protein structure. The interaction with water induces further continuous protein motions on a small scale. Water acts as a plasticizer of displacements, which couple to functional processes such as open-closed transitions and ligand exchange. PMID:15893505
Proton transfer via a transient linear water-molecule chain in a membrane protein
Gerwert, Klaus
Proton transfer via a transient linear water-molecule chain in a membrane protein Erik Freiera,1-resolution protein ground-state structures of proton pumps and channels have revealed internal protein-bound water. An illustration of the formation of a pro- tonated protein-bound water cluster that is actively involved in proton
Lipid ion channels and the role of proteins.
Mosgaard, Lars D; Heimburg, Thomas
In the absence of proteins, synthetic lipid membranes can display quantized conduction events for ions that are virtually indistinguishable from those of protein channels. The phenomenological similarities between typical conductances are striking: they are of equal order and show similar lifetime distributions and current histograms. They can include conduction bursts, flickering, and multistep conductance. Lipid channels can be gated by voltage and blocked by drugs. They respond to changes in lateral membrane tension and temperature. Thus, they behave like voltage-gated, temperature-gated, and mechano-sensitive protein channels, or like receptors. The similarity between lipid and protein channels poses an important problem for the interpretation of protein channel data. For example, the Hodgkin-Huxley theory for nerve pulse conduction requires a selective mechanism for the conduction of sodium and potassium ions. To this end, the lipid membrane must act both as a capacitor and as an insulator. Nonselective ion conductance by mechanisms other than the gated protein channels challenges the proposed mechanism for pulse propagation. Nevertheless, textbooks rarely describe the properties of the lipid membrane surrounding the proteins in their discussions of membrane models. These similarities lead to important questions: Do these similarities in lipid and protein channels result from a common mechanism, or are these similarities fortuitous? What distinguishes protein channels from lipid channels, if anything? In this Account, we document experimental and theoretical findings that show the similarity between lipid and protein channels. We discuss important cases where protein channel function strongly correlates with the properties of the lipid. Based on statistical thermodynamics simulations, we discuss how such correlations could come about. We suggest that proteins can act as catalysts for lipid channel formation and that this hypothesis can explain some of the unexplained correlations between protein and lipid membrane function. PMID:23902303
The aquaporin-Z water channel gene of Escherichia coli: Structure, organization and phylogeny
Giuseppe Calamita; Bettina Kempf; Kenneth E Rudd; Mķlanie Bonhivers; Susanne Kneip; William R Bishal; Erhard Bremer; Peter Agre
Aquaporin water channel proteins are found throughout the plant and animal kingdoms, but the first prokaryotic water channel gene, aqpZ, was only recently identified in wild type Escherichia coli (Calamita G et al (1995) J Biol Chem 270, 29063¢29066). Here we define the organization of aqpZ in E coli, produce the AqpZ protein and compare the AqpZ phylogeny to that
Water in protein structure prediction
Papoian, Garegin A.; Ulander, Johan; Eastwood, Michael P.; Luthey-Schulten, Zaida; Wolynes, Peter G.
Proteins have evolved to use water to help guide folding. A physically motivated, nonpairwise-additive model of water-mediated interactions added to a protein structure prediction Hamiltonian yields marked improvement in the quality of structure prediction for larger proteins. Free energy profile analysis suggests that long-range water-mediated potentials guide folding and smooth the underlying folding funnel. Analyzing simulation trajectories gives direct evidence that water-mediated interactions facilitate native-like packing of supersecondary structural elements. Long-range pairing of hydrophilic groups is an integral part of protein architecture. Specific water-mediated interactions are a universal feature of biomolecular recognition landscapes in both folding and binding. PMID:14988499
Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey
1. INTAKE CHANNEL LOOKING NORTHEAST; WATER FROM BEAVER BROOK ENTERS THE INTAKE CHANNEL HERE. - Hondius Water Line, 1.6 miles Northwest of Park headquarters building & 1 mile Northwest of Beaver Meadows entrance station, Estes Park, Larimer County, CO
Simulations of ion channels ¢ watching ions and water move
Mark S. P. Sansom; Indira H. Shrivastava; Kishani M. Ranatunga; Graham R. Smith
Ion channels mediate electrical excitability in neurons and muscle. Three-dimensional structures for model peptide channels and for a potassium (K+) channel have been combined with computer simulations to permit rigorous exploration of structure¢function relations of channels. Water molecules and ions within transbilayer pores tend to diffuse more slowly than in bulk solutions. In the narrow selectivity filter of the bacterial
Ion channel regulation by protein S-acylation
Protein S-acylation, the reversible covalent fatty-acid modification of cysteine residues, has emerged as a dynamic posttranslational modification (PTM) that controls the diversity, life cycle, and physiological function of numerous ligand- and voltage-gated ion channels. S-acylation is enzymatically mediated by a diverse family of acyltransferases (zDHHCs) and is reversed by acylthioesterases. However, for most ion channels, the dynamics and subcellular localization at which S-acylation and deacylation cycles occur are not known. S-acylation can control the two fundamental determinants of ion channel function: (1) the number of channels resident in a membrane and (2) the activity of the channel at the membrane. It controls the former by regulating channel trafficking and the latter by controlling channel kinetics and modulation by other PTMs. Ion channel function may be modulated by S-acylation of both pore-forming and regulatory subunits as well as through control of adapter, signaling, and scaffolding proteins in ion channel complexes. Importantly, cross-talk of S-acylation with other PTMs of both cysteine residues by themselves and neighboring sites of phosphorylation is an emerging concept in the control of ion channel physiology. In this review, I discuss the fundamentals of protein S-acylation and the tools available to investigate ion channel S-acylation. The mechanisms and role of S-acylation in controlling diverse stages of the ion channel life cycle and its effect on ion channel function are highlighted. Finally, I discuss future goals and challenges for the field to understand both the mechanistic basis for S-acylation control of ion channels and the functional consequence and implications for understanding the physiological function of ion channel S-acylation in health and disease. PMID:24821965
Activation of maxi-anion channel by protein tyrosine dephosphorylation.
Toychiev, Abduqodir H; Sabirov, Ravshan Z; Takahashi, Nobuyaki; Ando-Akatsuka, Yuhko; Liu, Hongtao; Shintani, Takafumi; Noda, Masaharu; Okada, Yasunobu
The maxi-anion channel with a large single-channel conductance of >300 pS, and unknown molecular identity, is functionally expressed in a large variety of cell types. The channel is activated by a number of experimental maneuvers such as exposing cells to hypotonic or ischemic stress. The most effective and consistent method of activating it is patch membrane excision. However, the activation mechanism of the maxi-anion channel remains poorly understood at present. In the present study, involvement of phosphorylation/dephosphorylation in excision-induced activation was examined. In mouse mammary fibroblastic C127 cells, activity of the channel was suppressed by intracellular application of Mg-ATP, but not Mg-5'-adenylylimidodiphosphate (AMP-PNP), in a concentration-dependent manner. When a cocktail of broad-spectrum tyrosine phosphatase inhibitors was applied, channel activation was completely abolished, whereas inhibitors of serine/threonine protein phosphatases had no effect. On the other hand, protein tyrosine kinase inhibitors brought the channel out of an inactivated state. In mouse adult skin fibroblasts (MAFs) in primary culture, similar maxi-anion channels were found to be activated on membrane excision, in a manner sensitive to tyrosine phosphatase inhibitors. In MAFs isolated from animals deficient in receptor protein tyrosine phosphatase (RPTP)zeta, activation of the maxi-anion channel was significantly slower and less prominent compared with that observed in wild-type MAFs; however, channel activation was restored by transfection of the RPTPzeta gene. Thus it is concluded that activation of the maxi-anion channel involves protein dephosphorylation mediated by protein tyrosine phosphatases that include RPTPzeta in mouse fibroblasts, but not in C127 cells. PMID:19657061
G protein modulation of recombinant P/Q-type calcium channels by regulators of G protein signalling proteins
Mark, Melanie D; Wittemann, Silke; Herlitze, Stefan
Fast synaptic transmission is triggered by the activation of presynaptic Ca2+ channels which can be inhibited by G?? subunits via G protein-coupled receptors (GPCR). Regulators of G protein signalling (RGS) proteins are GTPase-accelerating proteins (GAPs), which are responsible for >100-fold increases in the GTPase activity of G proteins and might be involved in the regulation of presynaptic Ca2+ channels. In this study we investigated the effects of RGS2 on G protein modulation of recombinant P/Q-type channels expressed in a human embryonic kidney (HEK293) cell line using whole-cell recordings. RGS2 markedly accelerates transmitter-mediated inhibition and recovery from inhibition of Ba2+ currents (IBa) through P/Q-type channels heterologously expressed with the muscarinic acetylcholine receptor M2 (mAChR M2). Both RGS2 and RGS4 modulate the prepulse facilitation properties of P/Q-type Ca2+ channels. G protein reinhibition is accelerated, while release from inhibition is slowed. These kinetics depend on the availability of G protein ? and ?? subunits which is altered by RGS proteins. RGS proteins unmask the Ca2+ channel ? subunit modulation of Ca2+ channel G protein inhibition. In the presence of RGS2, P/Q-type channels containing the ?2a and ?3 subunits reveal significantly altered kinetics of G protein modulation and increased facilitation compared to Ca2+ channels coexpressed with the ?1b or ?4 subunit. PMID:11018106
BIOCHEMISTRY: TRP Ion Channels--Two Proteins in One
NSDL National Science Digital Library
Irwin B. Levitan (University of Pennsylvania School of Medicine; Department of Neuroscience)
Access to the article is free, however registration and sign-in are required. It is well established that some proteins carry out more than one job in the cell, but so far, ion channels do not appear to exhibit this versatility. In their Perspective, Levitan and Cibulsky discuss a cluster of new findings showing that two members of the long TRP ion channel family, LTRPC7 and LTRPC2, are both ion channels and enzymes.
Studies of ion channels using expressed protein ligation.
Focke, Paul J; Valiyaveetil, Francis I
Expressed protein ligation (EPL) is a semisynthetic technique for the chemoselective ligation of a synthetic peptide to a recombinant peptide that results in a native peptide bond at the ligation site. EPL therefore allows us to engineer proteins with chemically defined, site-specific modifications. While EPL has been used mainly in investigations of soluble proteins, in recent years it has been increasingly used in investigations of integral membrane proteins. These include studies on the KcsA K(+) channel, the non-selective cation channel NaK, and the porin OmpF. These studies are discussed in this review. PMID:20965773
Iterative Water-filling for Gaussian Vector Multiple Access Channels
Li, Tiffany Jing
Iterative Water-filling for Gaussian Vector Multiple Access Channels W. Yu, W. Rhee, S. Boyd, and J. Cioffi Zhenlei Shen Lehigh University March 29, 2005 Zhenlei Shen (Lehigh) Iterative Water-filling for Gaussian Vector Multiple Access ChannelsMarch 29, 2005 1 / 13 #12;1 Quick Review 2 Iterative Water
X-ray structure of a protein-conducting channel
Bert van den Berg; William M. Clemons; Ian Collinson; Yorgo Modis; Enno Hartmann; Stephen C. Harrison; Tom A. Rapoport
A conserved heterotrimeric membrane protein complex, the Sec61 or SecY complex, forms a protein-conducting channel, allowing polypeptides to be transferred across or integrated into membranes. We report the crystal structure of the complex from Methanococcus jannaschii at a resolution of 3.2┼. The structure suggests that one copy of the heterotrimer serves as a functional translocation channel. The alpha-subunit has two
Water and polypeptide conformations in the gramicidin channel. A molecular dynamics study.
Chiu, S W; Subramaniam, S; Jakobsson, E; McCammon, J A
Theoretical studies of ion channels address several important questions. The mechanism of ion transport, the role of water structure, the fluctuations of the protein channel itself, and the influence of structural changes are accessible from these studies. In this paper, we have carried out a 70-ps molecular dynamics simulation on a model structure of gramicidin A with channel waters. The backbone of the protein has been analyzed with respect to the orientation of the carbonyl and the amide groups. The results are in conformity with the experimental NMR data. The structure of water and the hydrogen bonding network are also investigated. It is found that the water molecules inside the channel act as a collective chain; whereas the conformation in which all the waters are oriented with the dipoles pointing along the axis of the channel is a preferred one, others are also accessed during the dynamics simulation. A collective coordinate involving the channel waters and some of the hydrogen bonding peptide partners is required to describe the transition of waters from one configuration to the other. PMID:2476188
Water Binding in Whey Protein Concentrates
P. G. Kliman; B. A. Anderson; M. J. Pallansch
Measurements of the heat of fusion of free water in concentrated solutions of purified whey proteins showed that .5 g water\\/g whey protein would not freeze at --40 C. This water was defined as bound. Total bound water in protein solu- tions containing lactose and salts varied between .5 and 1.2 g water\\/g solids, with unfreezable water increasing as the
Water transport by the bacterial channel alpha-hemolysin
NASA Technical Reports Server (NTRS)
Paula, S.; Akeson, M.; Deamer, D.
This study is an investigation of the ability of the bacterial channel alpha-hemolysin to facilitate water permeation across biological membranes. alpha-Hemolysin channels were incorporated into rabbit erythrocyte ghosts at varying concentrations, and water permeation was induced by mixing the ghosts with hypertonic sucrose solutions. The resulting volume decrease of the ghosts was followed by time-resolved optical absorption at pH 5, 6, and 7. The average single-channel permeability coefficient of alpha-hemolysin for water ranged between 1.3x10-12 cm/s and 1.5x10-12 cm/s, depending on pH. The slightly increased single-channel permeability coefficient at lower pH-values was attributed to an increase in the effective pore size. The activation energy of water transport through the channel was low (Ea=5.4 kcal/mol), suggesting that the properties of water inside the alpha-hemolysin channel resemble those of bulk water. This conclusion was supported by calculations based on macroscopic hydrodynamic laws of laminar water flow. Using the known three-dimensional structure of the channel, the calculations accurately predicted the rate of water flow through the channel. The latter finding also indicated that water permeation data can provide a good estimate of the pore size for large channels.
Anoctamin and transmembrane channel-like proteins are evolutionarily related
Anoctamin (ANO) family of proteins, consisting of 10 members in mammals, are transmembrane proteins that have Ca2+-activated Cl- channel activities. Transmembrane channel-like (TMC) family of proteins, consisting of 8 members in mammals, are also transmembrane proteins of which mutations are implicated in various human conditions, such as hearing loss and epidermodysplasia verruciformis. Here we show that ANO and TMC proteins share high sequence similarity and probably the same membrane topology, indicating that these proteins are evolutionarily related. We found many conserved amino acid residues between the two families of proteins, especially in regions spanning the transmembrane domains TM1, TM4-TM5, and TM6-TM7. These findings imply that these proteins form one large family, which we term ANO/TMC superfamily and that TMC proteins may also function as channels for Cl- or possibly other ions. The ANO/TMC superfamily proteins are present in almost all the diverse groups of eukaryotic organisms, suggesting that the proteins function in important biological processes, such as ion homeostasis, in eukaryotic cells. PMID:19513534
Dual Channel Water Vapour Radiometer Development.
NASA Astrophysics Data System (ADS)
Stoew, S.; Rieck, C.
A new instrument for measuring the atmospheric water vapour is being developed at the Onsala Space Observatory. It is a dual channel microwave radiometer, measuring the sky brightness temperatures at the double-side bands centered at 21 GHz and 31.4 GHz respectively. Compared to the radiometer presently operating at the observatory, the new instrument is more compact and mobile. It will be used for comparisons at the Onsala site, especially studying the effect of narrower antenna beams. It will also be operated at other sites, e.g. in the Swedish GPS-networkSWEPOS. The authors will discuss the basic design which, for example, employes two conical horns equipped with lenses. Each channel uses two reference temperature black bodies (matched loads) for calibration: one "warm" - at stabilized room temperature and one "hot" which is regulated 100 degC. The stability tests on the latter showed confinement within +/- 0.03 degC around 100 degC. The recent work on the microwave part and the data acqisition (DAQ) system will be presented together with the solution for the positioning of the radiometer. The interaction between the control and DAQ system and the microwave block of the instrument will be discussedas well as the precision of the acquired data.
Capacity of MIMO Systems in Shallow Water Acoustic Channels
Stojanovic, Milica
Capacity of MIMO Systems in Shallow Water Acoustic Channels Andreja Radosevic, Dario Fertonani with the number of transmit or receive elements, whichever is smaller. The ergodic capacity of UWA MIMO channel on the information rate achievable through multiple-input multiple- output (MIMO) communications over UWA channels
Altered sodium channel-protein associations in critical illness myopathy
Background During the acute phase of critical illness myopathy (CIM) there is inexcitability of skeletal muscle. In a rat model of CIM, muscle inexcitability is due to inactivation of sodium channels. A major contributor to this sodium channel inactivation is a hyperpolarized shift in the voltage dependence of sodium channel inactivation. The goal of the current study was to find a biochemical correlate of the hyperpolarized shift in sodium channel inactivation. Methods The rat model of CIM was generated by cutting the sciatic nerve and subsequent injections of dexamethasone for 7?days. Skeletal muscle membranes were prepared from gastrocnemius muscles, and purification and biochemical analyses carried out. Immunoprecipitations were performed with a pan-sodium channel antibody, and the resulting complexes probed in Western blots with various antibodies. Results We carried out analyses of sodium channel glycosylation, phosphorylation, and association with other proteins. Although there was some loss of channel glycosylation in the disease, as assessed by size analysis of glycosylated and de-glycosylated protein in control and CIM samples, previous work by other investigators suggest that such loss would most likely shift channel inactivation gating in a depolarizing direction; thus such loss was viewed as compensatory rather than causative of the disease. A phosphorylation site at serine 487 was identified on the NaV 1.4 sodium channel ? subunit, but there was no clear evidence of altered phosphorylation in the disease. Co-immunoprecipitation experiments carried out with a pan-sodium channel antibody confirmed that the sodium channel was associated with proteins of the dystrophin associated protein complex (DAPC). This complex differed between control and CIM samples. Syntrophin, dystrophin, and plectin associated strongly with sodium channels in both control and disease conditions, while ?-dystroglycan and neuronal nitric oxide synthase (nNOS) associated strongly with the sodium channel only in CIM. Recording of action potentials revealed that denervated muscle in mice lacking nNOS was more excitable than control denervated muscle. Conclusion Taken together, these data suggest that the conformation/protein association of the sodium channel complex differs in control and critical illness myopathy muscle membranes; and suggest that nitric oxide signaling plays a role in development of muscle inexcitability. PMID:22935229
Water hardness influences Flavobacterium columnare pathogenesis in channel catfish
Technology Transfer Automated Retrieval System (TEKTRAN)
Studies were conducted to determine aspects of water chemistry responsible for large differences in pathogenesis and mortality rates in challenges of channel catfish Ictalurus punctatus with Flavobacterium columnare; challenges were conducted in water supplying the Stuttgart National Aquaculture Res...
Technology Transfer Automated Retrieval System (TEKTRAN)
This article presents a procedure to determine the characteristics of open water channels required for controller and filter design, with special focus on the resonance waves. Also, a new simplified model structure for open water channels is proposed. The procedure applies System Identification tool...
Biomimetic devices functionalized by membrane channel proteins
NASA Astrophysics Data System (ADS)
Schmidt, Jacob
We are developing a new family of active materials which derive their functional properties from membrane proteins. These materials have two primary components: the proteins and the membranes themselves. I will discuss our recent work directed toward development of a generic platform for a "plug-and-play" philosophy of membrane protein engineering. By creating a stable biomimetic polymer membrane a single molecular monolayer thick, we will enable the exploitation of the function of any membrane protein, from pores and pumps to sensors and energy transducers. Our initial work has centered on the creation, study, and characterization of the biomimetic membranes. We are attempting to make large areas of membrane monolayers using Langmuir-Blodgett film formation as well as through arrays of microfabricated black lipid membrane-type septa. A number of techniques allow the insertion of protein into the membranes. As a benchmark, we have been employing a model system of voltage-gated pore proteins, which have electrically controllable porosities. I will report on the progress of this work, the characterization of the membranes, protein insertion processes, and the yield and functionality of the composite.
Water sorption by proteins: Milk and whey proteins
John E. Kinsella; Patrick F. Fox; Louis B. Rockland
The content and physical state of water in foods influence their physical, chemical, quality, safety, and functional behavior. Information concerning the sorption behavior of dairy proteins, in the water activity (AW) range 0 to 0.9, is collated in this paper. The sorption behavior of proteins in general, the kinetics of absorption, factors affecting water binding, the phenomenon of desorption hysteresis,
S100 Calcium Binding Proteins and Ion Channels
Hermann, Anton; Donato, Rosario; Weiger, Thomas M.; Chazin, Walter J.
S100 Ca2+-binding proteins have been associated with a multitude of intracellular Ca2+-dependent functions including regulation of the cell cycle, cell differentiation, cell motility and apoptosis, modulation of membrane¢cytoskeletal interactions, transduction of intracellular Ca2+ signals, and in mediating learning and memory. S100 proteins are fine tuned to read the intracellular free Ca2+ concentration and affect protein phosphorylation, which makes them candidates to modulate certain ion channels and neuronal electrical behavior. Certain S100s are secreted from cells and are found in extracellular fluids where they exert unique extracellular functions. In addition to their neurotrophic activity, some S100 proteins modulate neuronal electrical discharge activity and appear to act directly on ion channels. The first reports regarding these effects suggested S100-mediated alterations in Ca2+ fluxes, K+ currents, and neuronal discharge activity. Recent reports revealed direct and indirect interactions with Ca2+, K+, Cl?, and ligand activated channels. This review focuses on studies of the physical and functional interactions of S100 proteins and ion channels. PMID:22539925
Water in channel-like cavities: structure and dynamics.
Sansom, M S; Kerr, I D; Breed, J; Sankararamakrishnan, R
Ion channels contain narrow columns of water molecules. It is of interest to compare the structure and dynamics of such intrapore water with those of the bulk solvent. Molecular dynamics simulations of modified TIP3P water molecules confined within channel-like cavities have been performed and the orientation and dynamics of the water molecules analyzed. Channels were modeled as cylindrical cavities with lengths ranging from 15 to 60 A and radii from 3 to 12 A. At the end of the molecular dynamics simulations water molecules were observed to be ordered into approximately concentric cylindrical shells. The waters of the outermost shell were oriented such that their dipoles were on average perpendicular to the normal of the wall of the cavity. Water dynamics were analyzed in terms of self-diffusion coefficients and rotational reorientation rates. For cavities of radii 3 and 6 A, water mobility was reduced relative to that of simulated bulk water. For 9- and 12-A radii confined water molecules exhibited mobilities comparable with that of the bulk solvent. If water molecules were confined within an hourglass-shaped cavity (with a central radius of 3 A increasing to 12 A at either end) a gradient of water mobility was observed along the cavity axis. Thus, water within simple models of transbilayer channels exhibits perturbations of structure and dynamics relative to bulk water. In particular the reduction of rotational reorientation rate is expected to alter the local dielectric constant within a transbilayer pore. Images FIGURE 6 PMID:8789086
Conductance and block of hair-cell mechanotransducer channels in transmembrane channel¢like protein mutants
Beurg, Maryline; Kim, Kyunghee X.
Transmembrane channel¢like (TMC) proteins TMC1 and TMC2 are crucial to the function of the mechanotransducer (MT) channel of inner ear hair cells, but their precise function has been controversial. To provide more insight, we characterized single MT channels in cochlear hair cells from wild-type mice and mice with mutations in Tmc1, Tmc2, or both. Channels were recorded in whole-cell mode after tip link destruction with BAPTA or after attenuating the MT current with GsMTx-4, a peptide toxin we found to block the channels with high affinity. In both cases, the MT channels in outer hair cells (OHCs) of wild-type mice displayed a tonotopic gradient in conductance, with channels from the cochlear base having a conductance (110 pS) nearly twice that of those at the apex (62 pS). This gradient was absent, with channels at both cochlear locations having similar small conductances, with two different Tmc1 mutations. The conductance of MT channels in inner hair cells was invariant with cochlear location but, as in OHCs, was reduced in either Tmc1 mutant. The gradient of OHC conductance also disappeared in Tmc1/Tmc2 double mutants, in which a mechanically sensitive current could be activated by anomalous negative displacements of the hair bundle. This ōreversed stimulus¢polarityö current was seen with two different Tmc1/Tmc2 double mutants, and with Tmc1/Tmc2/Tmc3 triple mutants, and had a pharmacological sensitivity comparable to that of native MT currents for most antagonists, except dihydrostreptomycin, for which the affinity was less, and for curare, which exhibited incomplete block. The existence in the Tmc1/Tmc2 double mutants of MT channels with most properties resembling those of wild-type channels indicates that proteins other than TMCs must be part of the channel pore. We suggest that an external vestibule of the MT channel may partly account for the channelÆs large unitary conductance, high Ca2+ permeability, and pharmacological profile, and that this vestibule is disrupted in Tmc mutants. PMID:24981230
Water-protein interactions from high-resolution protein crystallography.
Nakasako, Masayoshi
To understand the role of water in life at molecular and atomic levels, structures and interactions at the protein-water interface have been investigated by cryogenic X-ray crystallography. The method enabled a much clearer visualization of definite hydration sites on the protein surface than at ambient temperature. Using the structural models of proteins, including several hydration water molecules, the characteristics in hydration structures were systematically analysed for the amount, the interaction geometries between water molecules and proteins, and the local and global distribution of water molecules on the surface of proteins. The tetrahedral hydrogen-bond geometry of water molecules in bulk solvent was retained at the interface and enabled the extension of a three-dimensional chain connection of a hydrogen-bond network among hydration water molecules and polar protein atoms over the entire surface of proteins. Networks of hydrogen bonds were quite flexible to accommodate and/or to regulate the conformational changes of proteins such as domain motions. The present experimental results may have profound implications in the understanding of the physico-chemical principles governing the dynamics of proteins in an aqueous environment and a discussion of why water is essential to life at a molecular level. PMID:15306376
Regulation of ion channels by CAMP-dependent protein kinase and A-kinase anchoring proteins
Scott, John D.
-kinase anchoring protein AMPA a-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid C catalytic subunit Kca calcium to calcium channels and enhances their regulation in multiple cell types. Addresses *$Department
Interactions between Plasma Membrane Aquaporins Modulate Their Water Channel Activity
Fetter, Karolina; Van Wilder, Valķrie; Moshelion, Menachem; Chaumont, Franńois
Plant plasma membrane intrinsic proteins (PIPs) cluster in two evolutionary subgroups, PIP1 and PIP2, with different aquaporin activities when expressed in Xenopus oocytes. Maize ZmPIP1;1 and ZmPIP1;2 do not increase the osmotic water permeability coefficient (Pf), whereas ZmPIP2;1, ZmPIP2;4, and ZmPIP2;5 do. Here, we show that coexpression of the nonfunctional ZmPIP1;2 and the functional ZmPIP2;1, ZmPIP2;4, or ZmPIP2;5 resulted in an increase in Pf that was dependent on the amount of injected ZmPIP1;2 complementary RNA. Confocal analysis of oocytes expressing ZmPIP1;2¢green fluorescent protein (GFP) alone or ZmPIP1;2-GFP plus ZmPIP2;5 showed that the amount of ZmPIP1;2-GFP present in the plasma membrane was significantly greater in coexpressing cells. Nickel affinity chromatography purification of ZmPIP2;1 fused to a His tag coeluted with ZmPIP1;2-GFP demonstrated physical interaction and heteromerization of both isoforms. Interestingly, coexpression of ZmPIP1;1 and ZmPIP2;5 did not result in a greater increase in Pf than did the expression of ZmPIP2;5 alone, but coexpression of the ZmPIP1;1 and ZmPIP1;2 isoforms induced a Pf increase, indicating that PIP1 isoform heteromerization is required for both of them to act as functional water channels. Mutational analysis demonstrated the important role of the C-terminal part of loop E in PIP interaction and water channel activity induction. This study has revealed a new mechanism of plant aquaporin regulation that might be important in plant water relations. PMID:14671024
G Protein Regulation of Ion Channels and Abscisic Acid
Jones, Alan M.
G Protein Regulation of Ion Channels and Abscisic Acid Signaling in Arabidopsis Guard Cells Xi-Qing Wang,1 Hemayet Ullah,2 Alan M. Jones,2 Sarah M. Assmann1 * The phytohormone abscisic acid (ABA by inwardly rectifying K chan- nels. During inhibition of stomatal opening by the plant hormone abscisic acid
Water Transport in Hydrophilic Channels of Nafion (DMR 0819860)
Petta, Jason
Water Transport in Hydrophilic Channels of Nafion (DMR 0819860) Qiao Zhao, Paul Majsztrik and Jay, consists of sulfonic acid groups dispersed in a teflon-like hydrophobic matrix. It was shown that water of the sulfonic acids; (ii) (10%water forms a hydration shell and forms a hydrophilic percolation pathway
The Acoustical Channel the Transfer Function of Water Pipes
Henkel, Werner
The Acoustical Channel the Transfer Function of Water Pipes Supervisor Prof Werner Henkel Student in impure water, the ultrasound waves are the only means of communication through water for long distance. Severe study of underwater sound propagation dates back to the first and second world war. In that age
This study is on water temperature dynamics in the shallow field channels of the USEPA Monticello Ecological Research Station (MERS). The hydraulic and temperature environment in the MERS channels was measured and simulated to provide some background for several biological studie...
Upregulation of aquaporin 2 water channel expression in pregnant rats.
Ohara, M; Martin, P Y; Xu, D L; St John, J; Pattison, T A; Kim, J K; Schrier, R W
Water retention is characteristic of pregnancy but the mechanism(s) of the altered water metabolism has yet to be elucidated. The collecting duct water channel, aquaporin 2 (AQP2), plays a pivotal role in the renal water regulation, and we hypothesized that AQP2 expression could be modified during pregnancy. Sprague-Dawley female rats were studied on days 7 (P7), 14 (P14), and 20 (P20) of pregnancy, and expression of AQP2 in papillae was examined. Nonpregnant (NP) littermates were used as controls. Plasma osmolalities were significantly lower in pregnant rats by day 7 of gestation (P7 283.8+/-1.82, P14 284.3+/-1.64, P < 0.001, P20 282. 4+/-1.32, P < 0.0001, vs. NP 291.8+/-1.06 mosmol/kgH2O). However, plasma vasopressin concentrations in pregnant rats were not significantly different than in nonpregnant rats (NP 1.03+/-0.14, P7 1.11+/-0.21, P14 1.15+/-0.21, P20 1.36+/-0.24 pg/ml, NS). The mRNA of AQP2 was increased early during pregnancy: AQP2/beta actin: P7 196+/-17.9, P14 200+/-6.8, and P20 208+/-15.5%, P < 0.005 vs. NP (100+/-11.1%). AQP2 protein was also increased during pregnancy: AQP2 protein: P7 269+/-10.0, P14 251+/-12.0, P < 0.0001, and P20 250+/-13.6%, P < 0.001 vs. NP (100+/-12.5%). The effect of V2 vasopressin receptor antagonist, OPC-31260, was then investigated. AQP2 mRNA was suppressed significantly by OPC-31260 administration to P14 rats (AQP2/beta actin: P14 with OPC-31260 39.6+/-1.7%, P < 0.001 vs. P14 with vehicle) and was decreased to the same level of expression as NP rats receiving OPC-31260. Similar findings were found with the analysis of AQP2 protein. The decreased plasma osmolality of P14 rats was not modified by OPC-31260. The results of the study indicate that upregulation of AQP2 contributes to the water retention in pregnancy through a V2 receptor-mediated effect. In addition to vasopressin, other factors may be involved in this upregulation. PMID:9486978
FAITH Water Channel Flow Visualization - Duration: 56 seconds.
Water channel flow visualization experiments are performed on a three dimensional model of a small hill. This experiment was part of a series of measurements of the complex fluid flow around the hi...
Nanometer-scale water- and proton-diffusion heterogeneities across water channels in polymer electrolyte membranes.
Song, Jinsuk; Han, Oc Hee; Han, Songi
Nafion, the most widely used polymer for electrolyte membranes (PEMs) in fuel cells, consists of a fluorocarbon backbone and acidic groups that, upon hydration, swell to form percolated channels through which water and ions diffuse. Although the effects of the channel structures and the acidic groups on water/ion transport have been studied before, the surface chemistry or the spatially heterogeneous diffusivity across water channels has never been shown to directly influence water/ion transport. By the use of molecular spin probes that are selectively partitioned into heterogeneous regions of the PEM and Overhauser dynamic nuclear polarization relaxometry, this study reveals that both water and proton diffusivity are significantly faster near the fluorocarbon and the acidic groups lining the water channels than within the water channels. The concept that surface chemistry at the (sub)nanometer scale dictates water and proton diffusivity invokes a new design principle for PEMs. PMID:25630609
Activation of purified calcium channels by stoichiometric protein phosphorylation
Nunoki, K.; Florio, V.; Catterall, W.A. (Univ. of Washington, Seattle (USA))
Purified dihydropyridine-sensitive calcium channels from rabbit skeletal muscle were reconstituted into phosphatidylcholine vesicles to evaluate the effect of phosphorylation by cyclic AMP-dependent protein kinase (PK-A) on their function. Both the rate and extent of {sup 45}Ca{sup 2+} uptake into vesicles containing reconstituted calcium channels were increased severalfold after incubation with ATP and PK-A. The degree of stimulation of {sup 45}Ca{sup 2+} uptake was linearly proportional to the extent of phosphorylation of the alpha 1 and beta subunits of the calcium channel up to a stoichiometry of approximately 1 mol of phosphate incorporated into each subunit. The calcium channels activated by phosphorylation were determined to be incorporated into the reconstituted vesicles in the inside-out orientation and were completely inhibited by low concentrations of dihydropyridines, phenylalkylamines, Cd{sup 2+}, Ni{sup 2+}, and Mg{sup 2+}. The results demonstrate a direct relationship between PK-A-catalyzed phosphorylation of the alpha 1 and beta subunits of the purified calcium channel and activation of the ion conductance activity of the dihydropyridine-sensitive calcium channels.
Structural basis of water-specific transport through the AQP1 water channel.
Sui, H; Han, B G; Lee, J K; Walian, P; Jap, B K
Water channels facilitate the rapid transport of water across cell membranes in response to osmotic gradients. These channels are believed to be involved in many physiological processes that include renal water conservation, neuro-homeostasis, digestion, regulation of body temperature and reproduction. Members of the water channel superfamily have been found in a range of cell types from bacteria to human. In mammals, there are currently 10 families of water channels, referred to as aquaporins (AQP): AQP0-AQP9. Here we report the structure of the aquaporin 1 (AQP1) water channel to 2.2 A resolution. The channel consists of three topological elements, an extracellular and a cytoplasmic vestibule connected by an extended narrow pore or selectivity filter. Within the selectivity filter, four bound waters are localized along three hydrophilic nodes, which punctuate an otherwise extremely hydrophobic pore segment. This unusual combination of a long hydrophobic pore and a minimal number of solute binding sites facilitates rapid water transport. Residues of the constriction region, in particular histidine 182, which is conserved among all known water-specific channels, are critical in establishing water specificity. Our analysis of the AQP1 pore also indicates that the transport of protons through this channel is highly energetically unfavourable. PMID:11780053
The Stoichiometry of G Binding to G-protein-regulated Inwardly Rectifying K Channels (GIRKs)*
Clapham, David E.
The Stoichiometry of G Binding to G-protein-regulated Inwardly Rectifying K Channels (GIRKs, Boston, Massachusetts 02115 G-protein-coupled inwardly rectifying K (GIRK; Kir3.x) channels are the primary effectors of numerous G-protein-coupled receptors. GIRK channels decrease cellular excitability
Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey
5. GATE 5, INTAKE CHANNEL LOOKING SOUTH; WATER FROM GATE 5 ENTERED DITCH AND IRRIGATED HONDIUS' FIELDS. - Hondius Water Line, 1.6 miles Northwest of Park headquarters building & 1 mile Northwest of Beaver Meadows entrance station, Estes Park, Larimer County, CO
Model studies of dense water overflows in the Faroese Channels
NASA Astrophysics Data System (ADS)
Cuthbertson, Alan; Davies, Peter; Stashchuk, Nataliya; Vlasenko, Vasiliy
The overflow of dense water from the Nordic Seas through the Faroese Channel system was investigated through combined laboratory experiments and numerical simulations using the Massachusetts Institute of Technology General Circulation Model. In the experimental study, a scaled, topographic representation of the Faroe-Shetland Channel, Wyville-Thomson Basin and Ridge and Faroe Bank Channel seabed bathymetry was constructed and mounted in a rotating tank. A series of parametric experiments was conducted using dye-tracing and drogue-tracking techniques to investigate deep-water overflow pathways and circulation patterns within the modelled region. In addition, the structure of the outflowing dense bottom water was investigated through density profiling along three cross-channel transects located in the Wyville-Thomson Basin and the converging, up-sloping approach to the Faroe Bank Channel. Results from the dye-tracing studies demonstrate a range of parametric conditions under which dense water overflow across the Wyville-Thomson Ridge is shown to occur, as defined by the Burger number, a non-dimensional length ratio and a dimensionless dense water volume flux parameter specified at the Faroe-Shetland Channel inlet boundary. Drogue-tracking measurements reveal the complex nature of flow paths and circulations generated in the modelled topography, particularly the development of a large anti-cyclonic gyre in the Wyville-Thompson Basin and up-sloping approach to the Faroe Bank Channel, which diverts the dense water outflow from the Faroese shelf towards the Wyville-Thomson Ridge, potentially promoting dense water spillage across the ridge itself. The presence of this circulation is also indicated by associated undulations in density isopycnals across the Wyville-Thomson Basin. Numerical simulations of parametric test cases for the main outflow pathways and density structure in a similarly-scaled Faroese Channels model domain indicate excellent qualitative agreement with the experimental observations and measurements. In addition, the comparisons show that strong temporal variability in the predicted outflow pathways and circulations have a strong influence in regulating the Faroe Bank Channel and Wyville-Thomson Ridge overflows, as well as in determining the overall response in the Faroese Channels to changes in the Faroe-Shetland Channel inlet boundary conditions.
Nanosecond Relaxation Dynamics of Hydrated Proteins: Water versus protein contributions
Khodadadi, S [University of Akron; Curtis, J. E. [National Institute of Standards and Technology (NIST), Gaithersburg, MD; Sokolov, Alexei P [ORNL
We have studied picosecond to nanosecond dynamics of hydrated protein powders using dielectric spectroscopy and molecular dynamics (MD) simulations. Our analysis of hydrogen-atom single particle dynamics from MD simulations focused on main ( main tens of picoseconds) and slow ( slow nanosecond) relaxation processes that were observed in dielectric spectra of similar hydrated protein samples. Traditionally, the interpretation of these processes observed in dielectric spectra has been ascribed to the relaxation behavior of hydration water tightly bounded to a protein and not to protein atoms. Detailed analysis of the MD simulations and comparison to dielectric data indicate that the observed relaxation process in the nanosecond time range of hydrated protein spectra is mainly due to protein atoms. The relaxation processes involve the entire structure of protein including atoms in the protein backbone, side chains, and turns. Both surface and buried protein atoms contribute to the slow processes; however, surface atoms demonstrate slightly faster relaxation dynamics. Analysis of the water molecule residence and dipolar relaxation correlation behavior indicates that the hydration water relaxes at much shorter time scales.
Properties of underwater acoustic communication channels in shallow water.
Yang, T C
Underwater acoustic channels are band-limited and reverberant, posing many obstacles to reliable, phase-coherent acoustic communications. While many high frequency communication experiments have been conducted in shallow water, few have carried out systematic studies on the channel properties at a time scale relevant for communications. To aid communication system design, this paper analyzes at-sea data collected in shallow water under various conditions to illustrate how the ocean environments (sea surface waves and random ocean medium) can affect the signal properties. Channel properties studied include amplitude and phase variations, and temporal coherence of individual paths as well as the temporal and spatial coherence of multipaths at different time scales. Reasons for the coherence loss are hypothesized. PMID:22280578
The stream channel incision syndrome and water quality
Technology Transfer Automated Retrieval System (TEKTRAN)
Watershed development often triggers channel incision, which accounts for 60-90% of sediments leaving many disturbed watersheds. Impacts of such incision on water quality processes and the implication of such impairment on stream biota are relevant to issues associated with establishing total maxim...
Expression of Aquaporin Water Channels in Rat Taste Buds
Kristina J. Watson; Insook Kim; Arian F. Baquero; Catherine A. Burks; Lidong Liu; Timothy A. Gilbertson
In order to gain insight into the molecular mechanisms that allow taste cells to respond to changes in their osmotic environment, we have used primarily immunocytochemical and molecular approaches to look for evidence of the presence of aquaporin-like water channels in taste cells. Labeling of isolated taste buds from the fungiform, foliate, and vallate papillae in rat tongue with antibodies
Grand canonical Monte Carlo simulations of water in protein environments
NASA Astrophysics Data System (ADS)
Woo, Hyung-June; Dinner, Aaron R.; Roux, BenoŅt
The grand canonical simulation algorithm is considered as a general methodology to sample the configuration of water molecules confined within protein environments. First, the probability distribution of the number of water molecules and their configuration in a region of interest for biochemical simulations, such as the active site of a protein, is derived by considering a finite subvolume in open equilibrium with a large system serving as a bulk reservoir. It is shown that the influence of the bulk reservoir can be represented as a many-body potential of mean force acting on the atoms located inside the subvolume. The grand canonical Monte Carlo (GCMC) algorithm, augmented by a number of technical advances to increase the acceptance of insertion attempts, is implemented, and tested for simple systems. In particular, the method is illustrated in the case of a pure water box with periodic boundary conditions. In addition, finite spherical systems of pure water and containing a dialanine peptide, are simulated with GCMC while the influence of the surrounding infinite bulk is incorporated using the generalized solvent boundary potential [W. Im, S. BernĶche, and B. Roux, J. Chem. Phys. 114, 2924 (2001)]. As a last illustration of water confined in the interior of a protein, the hydration of the central cavity of the KcsA potassium channel is simulated.
Water rotation barriers on protein molecular surfaces
NASA Astrophysics Data System (ADS)
Tompa, K.; Bokor, M.; Verebķlyi, T.; Tompa, P.
The experimental characterization of hindered-rotation barriers and mapping the energetic heterogeneity of water molecules bound to the molecular "surface" of proteins is critical for understanding the functional interaction of proteins with their environment. Here, we show how to achieve this goal by an original wide-line NMR procedure, which is based on the spectral motional narrowing phenomenon following the melting (thawing) process of interfacial ice. The procedure highlights the differences between globular and intrinsically disordered proteins and it enables to delineate the effect of solvent on protein structure, making a distinction between point mutants, monomeric and oligomeric states, and characterizing the molecular interactions taking part in different cellular processes. We put this unique experimental approach introducing novel physical quantities and quantifying the heterogeneous distribution of motional activation energy of water in the interfacial landscape into a historical perspective, demonstrating its utility through a variety of globular and disordered proteins.
Dynamics of Protein Hydration Water
M. Wolf; S. Emmert; R. Gulich; P. Lunkenheimer; A. Loidl
We present the frequency- and temperature-dependent dielectric properties of lysozyme solutions in a broad concentration regime, measured at subzero temperatures and compare the results with measurements above the freezing point of water and on hydrated lysozyme powder. Our experiments allow examining the dynamics of unfreezable hydration water in a broad temperature range including the so-called No Man's Land (160 - 235 K). The obtained results prove the bimodality of the hydration shell dynamics and are discussed in the context of the highly-debated fragile-to-strong transition of water.
Channel Extension in Deep-Water Distributive Systems
NASA Astrophysics Data System (ADS)
Hoyal, D. C.; Sheets, B. A.
The cyclic nature of channel and lobe formation in submarine fans is the result of the unstable and ephemeral nature of newly formed distributary channels. Avulsion cycles are initiated as unconfined sheet flow immediately following avulsion followed by stages of channel incision and extension, deposition of channel mouth deposits, and often channel backfilling. In contrast with those in alluvial and deltaic environments, avulsion cycles in submarine fans are relatively poorly understood due to the difficulty of observing deep ocean processes, either over short timescales needed to measure the hydrodynamics of active turbidity currents, or over longer timescales needed for the morphodynamic evolution of individual distributary channels and avulsion events. Here we report the results of over 80 experiments in a 5m x 3m x1m deep tank using saline (NaCl) density flows carrying low-density plastic sediment (SG 1.5) flowing down an inclined ramp. These experiments were designed to investigate trends observed in earlier self-organized experimental submarine fans with well-developed avulsion cycles, in which distributive lobes were observed to form on relatively high slopes. In particular, we were interested in investigating the relationship between channel extension length (distance from the inlet to the point where the flow becomes de-channelized, transitioning into a mouth-bar/lobe) and slope. The results of the experiments are clear but counter-intuitive. Channels appear to extend in discrete segments and channel extension length is inversely related to slope over a wide range of slopes (5-17 degrees). In addition, channel extension seems largely independent of inlet flow density (salt concentration) over the experimental range (10-24 g/cc). Measurements of densimetric Froude number (Fr') indicate Fr' increases downstream to near critical conditions at the channel lobe transition. Our preliminary interpretation is that distributary channels become unstable due to acceleration to Fr'-critical conditions and the formation of a depositional hydraulic jump, which perturbs sediment transport and ends channel extension. Similar morphodynamic length scale controls are observed in shallow water fan-delta experiments (e.g., SAFL DB-03) and in 2-D depositional cyclic steps. The experiments seem to explain two interesting observations from the earlier self-organized fan experiments and from real submarine fans. Firstly, the observation of 'perched' fills at the steep entrances to salt withdrawal minibasins (e.g., in the Gulf of Mexico) suggesting higher sedimentation rates (or inefficient sediment transport) on higher slopes (initially higher than at the slope break downstream). Secondly, strong progradation as the fan evolves and slope decreases in 'perched' fans suggests increasing flow efficiency on lower slopes, at least over a certain window of parameter space. Apparently deep water systems have a tendency to self-regulate even when flows differ significantly in initial density. The observed modulation to Fr'-critical flow appears to be an important control on length scales in deep- water distributive channel systems, potentially explaining strong deepwater progradation or 'delta-like' patterns that have remained paradoxical. Near critical conditions have been inferred from observations of many active submarine fans but the extent to which these results from conservative density currents apply to non-conservative and potentially 'ignitive' turbidity currents is the subject of ongoing investigation.
Transmembrane Passage of Hydrophobic Compounds Through a Protein Channel Wall
Hearn, E.; Patel, D; Lepore, D; Indic, M; van den Berg, B
Membrane proteins that transport hydrophobic compounds have important roles in multi-drug resistance and can cause a number of diseases, underscoring the importance of protein-mediated transport of hydrophobic compounds. Hydrophobic compounds readily partition into regular membrane lipid bilayers, and their transport through an aqueous protein channel is energetically unfavourable3. Alternative transport models involving acquisition from the lipid bilayer by lateral diffusion have been proposed for hydrophobic substrates. So far, all transport proteins for which a lateral diffusion mechanism has been proposed function as efflux pumps. Here we present the first example of a lateral diffusion mechanism for the uptake of hydrophobic substrates by the Escherichia coli outer membrane long-chain fatty acid transporter FadL. A FadL mutant in which a lateral opening in the barrel wall is constricted, but which is otherwise structurally identical to wild-type FadL, does not transport substrates. A crystal structure of FadL from Pseudomonas aeruginosa shows that the opening in the wall of the {beta}-barrel is conserved and delineates a long, hydrophobic tunnel that could mediate substrate passage from the extracellular environment, through the polar lipopolysaccharide layer and, by means of the lateral opening in the barrel wall, into the lipid bilayer from where the substrate can diffuse into the periplasm. Because FadL homologues are found in pathogenic and biodegrading bacteria, our results have implications for combating bacterial infections and bioremediating xenobiotics in the environment.
Regulators of G Protein Signaling Attenuate the G Protein¢mediated Inhibition of N-Type Ca Channels
Melliti, Karim; Meza, Ulises; Fisher, Rory; Adams, Brett
Regulators of G protein signaling (RGS) proteins bind to the ? subunits of certain heterotrimeric G proteins and greatly enhance their rate of GTP hydrolysis, thereby determining the time course of interactions among G?, G??, and their effectors. Voltage-gated N-type Ca channels mediate neurosecretion, and these Ca channels are powerfully inhibited by G proteins. To determine whether RGS proteins could influence Ca channel function, we recorded the activity of N-type Ca channels coexpressed in human embryonic kidney (HEK293) cells with G protein¢coupled muscarinic (m2) receptors and various RGS proteins. Coexpression of full-length RGS3T, RGS3, or RGS8 significantly attenuated the magnitude of receptor-mediated Ca channel inhibition. In control cells expressing ?1B, ?2, and ?3 Ca channel subunits and m2 receptors, carbachol (1 ?M) inhibited whole-cell currents by ?80% compared with only ?55% inhibition in cells also expressing exogenous RGS protein. A similar effect was produced by expression of the conserved core domain of RGS8. The attenuation of Ca current inhibition resulted primarily from a shift in the steady state dose¢response relationship to higher agonist concentrations, with the EC50 for carbachol inhibition being ?18 nM in control cells vs. ?150 nM in RGS-expressing cells. The kinetics of Ca channel inhibition were also modified by RGS. Thus, in cells expressing RGS3T, the decay of prepulse facilitation was slower, and recovery of Ca channels from inhibition after agonist removal was faster than in control cells. The effects of RGS proteins on Ca channel modulation can be explained by their ability to act as GTPase-accelerating proteins for some G? subunits. These results suggest that RGS proteins may play important roles in shaping the magnitude and kinetics of physiological events, such as neurosecretion, that involve G protein¢modulated Ca channels. PMID:9874691
NMR Structure and Ion Channel Activity of the p7 Protein from Hepatitis C Virus*
Montserret, Roland; Saint, Nathalie; Vanbelle, Christophe; Salvay, Andrķs Gerardo; Simorre, Jean-Pierre; Ebel, Christine; Sapay, Nicolas; Renisio, Jean-Guillaume; B÷ckmann, Anja; Steinmann, Eike; Pietschmann, Thomas; Dubuisson, Jean; Chipot, Christophe; Penin, Franńois
The small membrane protein p7 of hepatitis C virus forms oligomers and exhibits ion channel activity essential for virus infectivity. These viroporin features render p7 an attractive target for antiviral drug development. In this study, p7 from strain HCV-J (genotype 1b) was chemically synthesized and purified for ion channel activity measurements and structure analyses. p7 forms cation-selective ion channels in planar lipid bilayers and at the single-channel level by the patch clamp technique. Ion channel activity was shown to be inhibited by hexamethylene amiloride but not by amantadine. Circular dichroism analyses revealed that the structure of p7 is mainly ?-helical, irrespective of the membrane mimetic medium (e.g. lysolipids, detergents, or organic solvent/water mixtures). The secondary structure elements of the monomeric form of p7 were determined by 1H and 13C NMR in trifluoroethanol/water mixtures. Molecular dynamics simulations in a model membrane were combined synergistically with structural data obtained from NMR experiments. This approach allowed us to determine the secondary structure elements of p7, which significantly differ from predictions, and to propose a three-dimensional model of the monomeric form of p7 associated with the phospholipid bilayer. These studies revealed the presence of a turn connecting an unexpected N-terminal ?-helix to the first transmembrane helix, TM1, and a long cytosolic loop bearing the dibasic motif and connecting TM1 to TM2. These results provide the first detailed experimental structural framework for a better understanding of p7 processing, oligomerization, and ion channel gating mechanism. PMID:20667830
The influence of water on protein properties.
Mallamace, Francesco; Baglioni, Piero; Corsaro, Carmelo; Chen, Sow-Hsin; Mallamace, Domenico; Vasi, Cirino; Stanley, H Eugene
The "dynamic" or "glass" transition in biomolecules is as important to their functioning as the folding process. This transition occurs in the low temperature regime and has been related to the onset of biochemical activity that is dependent on the hydration level. This protein transition is believed to be triggered by the strong hydrogen bond coupling in the hydration water. We study the vibrational bending mode and measure it using Fourier Transform Infrared spectroscopy. We demonstrate that at the molecular level the hydration water bending mode bonds the C=O and N-H peptide groups, and find that the temperature of the "dynamic" protein transition is the same as the fragile-to-strong dynamic transition in confined water. The fragile-to-strong dynamic transition in water governs the nature of the H bonds between water and peptides and appears to be universal in supercooled glass-forming liquids. PMID:25362345
The influence of water on protein properties
NASA Astrophysics Data System (ADS)
Mallamace, Francesco; Baglioni, Piero; Corsaro, Carmelo; Chen, Sow-Hsin; Mallamace, Domenico; Vasi, Cirino; Stanley, H. Eugene
The "dynamic" or "glass" transition in biomolecules is as important to their functioning as the folding process. This transition occurs in the low temperature regime and has been related to the onset of biochemical activity that is dependent on the hydration level. This protein transition is believed to be triggered by the strong hydrogen bond coupling in the hydration water. We study the vibrational bending mode and measure it using Fourier Transform Infrared spectroscopy. We demonstrate that at the molecular level the hydration water bending mode bonds the C=O and N-H peptide groups, and find that the temperature of the "dynamic" protein transition is the same as the fragile-to-strong dynamic transition in confined water. The fragile-to-strong dynamic transition in water governs the nature of the H bonds between water and peptides and appears to be universal in supercooled glass-forming liquids.
Identification of a multiprotein "motor" complex binding to water channel aquaporin-2.
Noda, Yumi; Horikawa, Saburo; Katayama, Yoshifumi; Sasaki, Sei
Targeted positioning of water channel aquaporin-2 (AQP2) strictly regulates body water homeostasis. Trafficking of AQP2 to the apical membrane is critical to the reabsorption of water in renal collecting ducts. Recently, we have identified for the first time proteins which directly bind to AQP2: SPA-1, a GTPase-activating protein for Rap1, and cytoskeletal protein actin. Based on these findings, we have speculated the existence of a multiprotein complex which includes AQP2, SPA-1, and actin, for providing the mechanism which generates force and motion in AQP2 trafficking. To clarify the proteins comprising the complex, a large amount of AQP2-associated protein complex was isolated from the extract of rat kidney papilla using immunoaffinity column coupled with anti-AQP2 antibody and was analyzed by matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI-TOF MS). In addition to SPA-1 and actin, 11 proteins were identified using this method: ionized calcium binding adapter molecule 2, myosin regulatory light chain smooth muscle isoforms 2-A and 2-B, alpha-tropomyosin 5b, annexin A2 and A6, scinderin, gelsolin, alpha-actinin 4, alpha-II spectrin, and myosin heavy chain nonmuscle type A. Our findings show for the first time an AQP2-binding multiprotein "force generator" complex. This multiprotein complex may provide the machinery of driving AQP2 movement. PMID:15823548
Topological Predictions for Integral Membrane Channel and Carrier Proteins
Abhinay, Reddy; Jaehoon, Cho; Sam, Ling; Vamsee, Reddy; Maksim, Shlykov; Milton, Saier
We evaluated topological predictions for nine different programs, HMMTOP, TMHMM, SVMTOP, DAS, SOSUI, TOPCONS, PHOBIUS, MEMSAT-SVM (hereinafter referred to as MEMSAT), and SPOCTOPUS. These programs were first evaluated using four large topologically well-defined families of secondary transporters, and the three best programs were further evaluated using topologically more diverse families of channels and carriers. In the initial studies, the order of accuracy was: SPOCTOPUS>MEMSAT>HMMTOP>TOPCONS>PHOBIUS>TMHMM>SVMTOP>DAS>S OSUI. Some families, such as the Sugar Porter family (2.A.1.1) of the Major Facilitator Superfamily (MFS; TC# 2.A.1) and the Amino acid/Polyamine/Organocation (APC) Family (TC# 2.A.3), were correctly predicted with high accuracy while others, such as the Mitochondrial Carrier (MC) (TC# 2.A.29) and the K+ transporter (Trk) families (TC# 2.A.38), were predicted with much lower accuracy. For small, topologically homogeneous families, SPOCTOPUS and MEMSAT were generally most reliable, while with large, more diverse superfamilies, HMMTOP often proved to have the greatest prediction accuracy. We next developed a novel program, TM-STATS, that tabulates HMMTOP, SPOCTOPUS or MEMSAT-based topological predictions for any subdivision (class, subclass, superfamily, family, subfamily, or any combination of these) of the Transporter Classification Database (TCDB; www.tcdb.org) and examined the following subclasses: ?-type channel proteins (TC subclasses 1.A and 1.E), secreted poreforming toxins (TC subclass 1.C) and secondary carriers (subclass 2.A). Histograms 3 were generated for each of these subclasses, and the results were analyzed according to subclass, family and protein. The results provide an update of topological predictions for integral membrane transport proteins as well as guides for the development of more reliable topological prediction programs, taking family-specific characteristics into account. PMID:24992992
No Mystery! Water Carved the Outflow Channels on Mars
NASA Astrophysics Data System (ADS)
Coleman, N.
The enormous outflow channels of Chryse Planitia provide the best evidence that large amounts of water were once released onto the martian surface. The role of water has recently been challenged by the White Mars hypothesis, which claims that the channels were cut by CO2 gas-supported debris flows that also resurfaced the northern plains. Hoffman [Icarus, 2000] refers to a volumetric "misfit" between outburst channels and the chaos source zones. He explains that chaos collapse "...involves regolith alone which generates its own fluids from liquid CO2 and CO2-bearing ices within its own volume." Hoffman [LPSC 32, #1257] argues that release of liquid CO2 produced Aromatum Chaos, and a hypothetical energetic "jet" of gas and debris carved Ravi Vallis. He notes that water would have had to be locally recharged in many episodes to provide enough discharge to form the chaos and channel. However, these assertions appear incorrect because the fluid source was a distant surface impoundment, not local recharge. Carr [Water on Mars, 1996] describes a 400-km-long zone of subsidence that extends northward from Ganges Chasma to the source of Shalbatana Vallis. MOLA data reveal that this subsidence also extends eastward to Aromatum Chaos, the source of Ravi Vallis. The field relations show that a liquid-filled impoundment in Ganges Chasma drained northward via subterranean flowpaths to maintain surface flows in Shalbatana and Ravi Valles. The fact that the flows began at a surface impoundment virtually eliminates liquid CO2 as the flowing agent. Liquid CO2 would not be stable at the surface unless the atmospheric pressure exceeded 5 atm. A recent study by Stewart and Nimmo [JGR, in press] suggests that CO2 in liquid, solid, or clathrate form could not be preserved within the crust over geologic time. Liquid water is much closer to its stability field even on present-day Mars. Large outflow channels, such as Kasei and Tiu-Simud Valles, likely formed through the release of floodwaters dammed by ice and debris, analogous to the scabland flooding of eastern Washington. The water sources were probably ice-covered impoundments in ancestral Valles Marineris canyons. Subice volcanism was a possible source of heat to create liquid water. The former existence of transient water bodies near the surface can help to calibrate models of a volcanic-hydrologic climax during the Hesperian.
Balanced Protein-Water Interactions Improve Properties of Disordered Proteins and Non-Specific Protein Association.
Best, Robert B; Zheng, Wenwei; Mittal, Jeetain
Some frequently encountered deficiencies in all-atom molecular simulations, such as nonspecific protein-protein interactions being too strong, and unfolded or disordered states being too collapsed, suggest that proteins are insufficiently well solvated in simulations using current state-of-the-art force fields. To address these issues, we make the simplest possible change, by modifying the short-range protein-water pair interactions, and leaving all the water-water and protein-protein parameters unchanged. We find that a modest strengthening of protein-water interactions is sufficient to recover the correct dimensions of intrinsically disordered or unfolded proteins, as determined by direct comparison with small-angle X-ray scattering (SAXS) and F÷rster resonance energy transfer (FRET) data. The modification also results in more realistic protein-protein affinities, and average solvation free energies of model compounds which are more consistent with experiment. Most importantly, we show that this scaling is small enough not to affect adversely the stability of the folded state, with only a modest effect on the stability of model peptides forming ?-helix and ?-sheet structures. The proposed adjustment opens the way to more accurate atomistic simulations of proteins, particularly for intrinsically disordered proteins, protein-protein association, and crowded cellular environments. PMID:25400522
Evidence for water-permeable channels in auditory hair cells in the leopard frog.
Miller, Mia E; Nasiri, Arian K; Farhangi, Peyman O; Farahbakhsh, Nasser A; Lopez, Ivan A; Narins, Peter M; Simmons, Dwayne D
Auditory hair cells in the amphibian papilla (APHCs) of the leopard frog, Rana pipiens pipiens, have a significantly higher permeability to water than that observed in mammalian hair cells. The insensitivity of water permeability in frog hair cells to extracellular mercury suggests that an amphibian homologue of the water channel aquaporin-4 (AQP4) may mediate water transport in these cells. Using immunocytochemistry, we show that an AQP4-like protein is found in APHCs. Rabbit anti-AQP4 antibody was used in multiple-immunohistochemical staining experiments along with AP hair cell and hair bundle markers in leopard frog and mouse tissue. AQP4 immunoreactivity was found in the basal and apical poles of the APHCs and shows uniform immunoreactivity. This study provides the first identification and localization of an AQP4-like protein in the amphibian inner ear. We also report a more direct measure of hyperosmotically-induced volume changes in APHCs that confirms previous findings. The presence of water channels in anuran APHCs constitutes a novel physiological difference between amphibian and mammalian hair cell structure and function. PMID:22940201
Priorities in the discovery of the implications of water channels in epilepsy and duchenne muscular dystrophy.
Benga, I
In addition to the priority in the discovery of the first water channel protein in the red blood cell membrane the group of Gheorghe Benga in Cluj-Napoca, Romania, also has a world priority in the discovery of the implications of water channel proteins in epilepsy and Duchenne muscular dystrophy. This priority is briefly presented here. In 1977 Benga and Morariu reported a decreased water permeability of red blood cells in children with idiopathic epilepsy (cases selected by Ileana Benga). This investigation was performed as part of a program of research of hydroelectrolytic alterations in child epilepsy. On the other hand the group of Gheorghe Benga has reported a decreased water permeability of RBC in patients with Duchenne muscular dystrophy. These findings were interpreted as an expression of generalized membrane defects affecting water permeability in epilepsy and Duchenne muscular dystrophy. In recent years this idea was confirmed by reports indicating aquaporin abnormalities in the brain of epileptic patients and in the muscle of Duchenne muscular dystrophy patients. PMID:17543221
The role of transmembrane channel¢like proteins in the operation of hair cell mechanotransducer channels
Kim, Kyunghee X.; Beurg, Maryline; Hackney, Carole M.; Furness, David N.; Mahendrasingam, Shanthini
Sound stimuli elicit movement of the stereocilia that make up the hair bundle of cochlear hair cells, putting tension on the tip links connecting the stereocilia and thereby opening mechanotransducer (MT) channels. Tmc1 and Tmc2, two members of the transmembrane channel¢like family, are necessary for mechanotransduction. To assess their precise role, we recorded MT currents elicited by hair bundle deflections in mice with null mutations of Tmc1, Tmc2, or both. During the first postnatal week, we observed a normal MT current in hair cells lacking Tmc1 or Tmc2; however, in the absence of both isoforms, we recorded a large MT current that was phase-shifted 180░, being evoked by displacements of the hair bundle away from its tallest edge rather than toward it as in wild-type hair cells. The anomalous MT current in hair cells lacking Tmc1 and Tmc2 was blocked by FM1-43, dihydrostreptomycin, and extracellular Ca2+ at concentrations similar to those that blocked wild type. MT channels in the double knockouts carried Ca2+ with a lower permeability than wild-type or single mutants. The MT current in double knockouts persisted during exposure to submicromolar Ca2+, even though this treatment destroyed the tip links. We conclude that the Tmc isoforms do not themselves constitute the MT channel but are essential for targeting and interaction with the tip link. Changes in the MT conductance and Ca2+ permeability observed in the absence of Tmc1 mutants may stem from loss of interaction with protein partners in the transduction complex. PMID:24127526
Some thermodynamical aspects of protein hydration water
NASA Astrophysics Data System (ADS)
Mallamace, Francesco; Corsaro, Carmelo; Mallamace, Domenico; Vasi, Sebastiano; Vasi, Cirino; Stanley, H. Eugene; Chen, Sow-Hsin
We study by means of nuclear magnetic resonance the self-diffusion of protein hydration water at different hydration levels across a large temperature range that includes the deeply supercooled regime. Starting with a single hydration shell (h = 0.3), we consider different hydrations up to h = 0.65. Our experimental evidence indicates that two phenomena play a significant role in the dynamics of protein hydration water: (i) the measured fragile-to-strong dynamic crossover temperature is unaffected by the hydration level and (ii) the first hydration shell remains liquid at all hydrations, even at the lowest temperature.
Some thermodynamical aspects of protein hydration water.
Mallamace, Francesco; Corsaro, Carmelo; Mallamace, Domenico; Vasi, Sebastiano; Vasi, Cirino; Stanley, H Eugene; Chen, Sow-Hsin
We study by means of nuclear magnetic resonance the self-diffusion of protein hydration water at different hydration levels across a large temperature range that includes the deeply supercooled regime. Starting with a single hydration shell (h = 0.3), we consider different hydrations up to h = 0.65. Our experimental evidence indicates that two phenomena play a significant role in the dynamics of protein hydration water: (i) the measured fragile-to-strong dynamic crossover temperature is unaffected by the hydration level and (ii) the first hydration shell remains liquid at all hydrations, even at the lowest temperature. PMID:26049527
Aquaporins: highly regulated channels controlling plant water relations.
Chaumont, Franńois; Tyerman, Stephen D
Plant growth and development are dependent on tight regulation of water movement. Water diffusion across cell membranes is facilitated by aquaporins that provide plants with the means to rapidly and reversibly modify water permeability. This is done by changing aquaporin density and activity in the membrane, including posttranslational modifications and protein interaction that act on their trafficking and gating. At the whole organ level aquaporins modify water conductance and gradients at key "gatekeeper" cell layers that impact on whole plant water flow and plant water potential. In this way they may act in concert with stomatal regulation to determine the degree of isohydry/anisohydry. Molecular, physiological, and biophysical approaches have demonstrated that variations in root and leaf hydraulic conductivity can be accounted for by aquaporins but this must be integrated with anatomical considerations. This Update integrates these data and emphasizes the central role played by aquaporins in regulating plant water relations. PMID:24449709
Smoothed Particle Hydrodynamics for water wave propagation in a channel
NASA Astrophysics Data System (ADS)
Omidvar, Pourya; Norouzi, Hossein; Zarghami, Ahad
In this paper, Smoothed Particle Hydrodynamics (SPH) is used to simulate the propagation of waves in an intermediate depth water channel. The major advantage of using SPH is that no special treatment of the free surface is required, which is advantageous for simulating highly nonlinear flows with possible wave breaking. The SPH method has an option of different formulations with their own advantages and drawbacks to be implemented. Here, we apply the classical and Arbitrary Lagrange-Euler (ALE) formulation for wave propagation in a water channel. The classical SPH should come with an artificial viscosity which stabilizes the numerical algorithm and increases the accuracy. Here, we will show that the use of classical SPH with an artificial viscosity may cause the waves in the channel to decay. On the other hand, we will show that using the ALE-SPH algorithm with a Riemann solver is more stable, and in addition to producing the pressure fields with much less numerical noise, the waves propagate in the channel without dissipation.
A flow through water system for channel catfish fingerling cultureĀ
Steinbach, Donny W
. Stocking Procedure The number of fry used in the 1975 study were estimated by water displacement. Water displacement by 10 g of fry was determined and the number of fish counted. This relationship was then used to estimate numbers of fry by displacement... the biomass in the drums reached levels of 1184. 83 g/250 drum to 6441. 56 g/2000 drum (Tables 3, 4). At this time, size Table 2. Stocking and harvest data for the channel catfish fry in the 1976 culture experiment. Number fish stocked (estimsted) Average...
Protein Surface Recognition by Rational Design: Nanomolar Ligands for Potassium Channels
Trauner, Dirk
Protein Surface Recognition by Rational Design: Nanomolar Ligands for Potassium Channels Stefan N May 14, 2003; E-mail: email@example.com Potassium channels are among the core features regulation. Recently, the three-dimensional structures of the prokaryotic potassium channels KcsA3 and MthK,4
Identification of characteristic protein folding channels in a coarse-grained hydrophobic-polar peptide model
Bachmann, Michael
Identification of characteristic protein folding channels in a coarse-grained hydrophobic of protein folding is one of the major challenges of modern interdisciplinary science. Proteins are linear simulations of protein folding are difficult, mainly for two reasons. Firstly, the folding process is so slow
Slow vacuolar channels from barley mesophyll cells are regulated by 14-3-3 proteins
Sch÷nknecht, Gerald
Slow vacuolar channels from barley mesophyll cells are regulated by 14-3-3 proteins Paul W.J. van rights reserved. Key words: 14-3-3 protein; Barley mesophyll; Patch-clamp; Plant vacuole; Slow activating vacuolar Ca2 decreases single channel cur- rent in barley mesophyll vacuoles [7]. In Fava bean guard cell
Fiber optic multi-channel protein detector for use in preparative continuous annular chromatography
Athanasios Apostolidis; Hartmut Lehmann; G³nter Schwotzer; Reinhardt Willsch; Albert Prior; J³rgen Wolfgang; Ingo Klimant; Otto S Wolfbeis
Continuous annular chromatography is an effective method in the separation of preparative scale quantities of biological compounds including proteins where established batch chromatography borders on it. The need for identification or quantification of proteins triggered the development of respective detection units. Here, we describe two types of optical multi-channel detectors. The first is a fiber optic multi-channel detector suitable for
Effects of water temperature and dissolved oxygen on daily feed consumption, feed utilization and growth of channel catfish ( Ictalurus punctatus)
J. Alejandro Buentello; Delbert M Gatlin; William H Neill
Feed intake (FI), feed efficiency (FE), protein efficiency ratio (PER) and weight gain (WG) of juvenile channel catfish initially weighing 15.0▒0.23 g (10¢12 cm initial total length) were evaluated under three regimes of time-varying water temperature (mean daily water temperature for Stoneville, MS, USA; mean +3; and, mean ?3░C) and three of dissolved oxygen (DO; 100, 70 and 30% air
Molecular mechanism of H+ conduction in the single-file water chain of the gramicidin channel.
PomĶs, Rķgis; Roux, BenoŅt
The conduction of protons in the hydrogen-bonded chain of water molecules (or "proton wire") embedded in the lumen of gramicidin A is studied with molecular dynamics free energy simulations. The process may be described as a "hop-and-turn" or Grotthuss mechanism involving the chemical exchange (hop) of hydrogen nuclei between hydrogen-bonded water molecules arranged in single file in the lumen of the pore, and the subsequent reorganization (turn) of the hydrogen-bonded network. Accordingly, the conduction cycle is modeled by two complementary steps corresponding respectively to the translocation 1) of an ionic defect (H+) and 2) of a bonding defect along the hydrogen-bonded chain of water molecules in the pore interior. The molecular mechanism and the potential of mean force are analyzed for each of these two translocation steps. It is found that the mobility of protons in gramicidin A is essentially determined by the fine structure and the dynamic fluctuations of the hydrogen-bonded network. The translocation of H+ is mediated by spontaneous (thermal) fluctuations in the relative positions of oxygen atoms in the wire. In this diffusive mechanism, a shallow free-energy well slightly favors the presence of the excess proton near the middle of the channel. In the absence of H+, the water chain adopts either one of two polarized configurations, each of which corresponds to an oriented donor-acceptor hydrogen-bond pattern along the channel axis. Interconversion between these two conformations is an activated process that occurs through the sequential and directional reorientation of water molecules of the wire. The effect of hydrogen-bonding interactions between channel and water on proton translocation is analyzed from a comparison to the results obtained previously in a study of model nonpolar channels, in which such interactions were missing. Hydrogen-bond donation from water to the backbone carbonyl oxygen atoms lining the pore interior has a dual effect: it provides a coordination of water molecules well suited both to proton hydration and to high proton mobility, and it facilitates the slower reorientation or turn step of the Grotthuss mechanism by stabilizing intermediate configurations of the hydrogen-bonded network in which water molecules are in the process of flipping between their two preferred, polarized states. This mechanism offers a detailed molecular model for the rapid transport of protons in channels, in energy-transducing membrane proteins, and in enzymes. PMID:11964221
The Mechanism of Proton Exclusion in the Aquaporin-1 Water Channel
de Groot, Bert
) ions together with water 0022-2836/$ - see front matter q 2003 Elsevier Ltd. All rights reserved. EThe Mechanism of Proton Exclusion in the Aquaporin-1 Water Channel Bert L. de Groot1 *, Tomaso water channels. Remarkably, proton permeation is fully blocked, in contrast to most other water
Suppression of aquaporin, a mediator of water channel control in the carcinogenic liver fluke, Opisthorchis viverrini
Background Opisthorchiasis and Opisthorchis viverrini-associated bile duct cancer represent major public health threats in Thailand and Laos. The tegument of this food borne fluke plays pivotal roles in parasite metabolism, homeostasis and osmoregulation. Excretory/secretory products also pass from the fluke to the biliary environment, products that likely underlie pathogenesis of liver fluke infection. Aquaporins (AQPs), belong to the major intrinsic protein superfamily of integral plasma membrane channel proteins that selectively transport water across cell membranes. AQPs play key roles as water and ion transport channels through the tegument of helminth parasites. Methods Here, two forms of AQP mRNAs from the adult developmental stage of O. viverrini, termed O. viverrini aquaporin-1 and -2 (Ov-aqp-1 and -2) were investigated. Roles of Ov-aqp-1 and -2 in the movement of water across the tegument of this carcinogenic liver fluke were investigated using RNA interference. Results Ov-AQP-1 and Ov-AQP-2 contain unique characteristic asparagine-proline-alanine (NPA) motifs of AQP transmembrane proteins. Phylogenetic analysis indicated that Ov-AQPs belong to an expanding group of aquaglyceroporin-like water channel proteins characterized from helminth and protozoan parasites, which is pivotal to the specialized requirements of water and solute control during parasitism. Elevated transcription of Ov-aqp-1 was evident in the egg, cercaria, metacercaria and adult stages of O. viverrini, whereas Ov-aqp-2 transcripts were detected at higher level in egg, metacercaria, cercaria and adult stage, respectively. RNA interference using electroporated dsRNA suppressed transcript levels of Ov-aqp-1 and Ov-aqp-2 in adult worms by 58-99% over periods of up to 16Ādays in vitro. Suppression of Ov-aqp-1 and Ov-aqp-2 in vitro disabled water transport in adult flukes. Conclusion The apparently pivotal roles of Ov-AQP in solute homeostasis at the fluke surface suggest that deeper investigation will be informative for the pathophysiology of O. viverrini, and may uncover intervention targets, particularly in view of the singularly notable predilection of this pathogen for residence within ducts of the biliary tree. PMID:24885060
Selectivity of Protein Ion Channels and the Role of Buried Charges. Analytical Solutions, Numerical Calculations, and MD Simulations.
GarcĒa-Gimķnez, Elena; Alcaraz, Antonio; Aguilella-Arzo, Marcel; Aguilella, Vicente M
The preference of large protein ion channels for cations or anions is mainly determined by the electrostatic interactions of mobile ions with charged residues of the protein. Here we discuss the widely spread paradigm that the charges determining the channel selectivity are only those that can be considered solvent-accessible because of their location near the permeation pathways of ions and water molecules. Theoretical predictions for the electric potential and average ion densities inside the pore are presented using several approaches of increasing resolution: from analytical and numerical solutions of electrostatic equations in a model channel up to all-atom molecular dynamics simulations and continuum electrostatic calculations performed in a particular biological channel, the bacterial porin OmpF. The results highlight the role of protein dieletric properties and the importance of the initial choice of the residue ionization states in the understanding of the molecular basis of large channel selectivity irrespective of the level of resolution of the computational approach used. PMID:26091047
How protein surfaces induce anomalous dynamics of hydration water.
Pizzitutti, Francesco; Marchi, Massimo; Sterpone, Fabio; Rossky, Peter J
Water around biomolecules slows down with respect to pure water, and both rotation and translation exhibit anomalous time dependence in the hydration shell. The origin of such behavior remains elusive. We use molecular dynamics simulations of water dynamics around several designed protein models to establish the connection between the appearance of the anomalous dynamics and water-protein interactions. For the first time we quantify the separate effect of protein topological and energetic disorder on the hydration water dynamics. When a static protein structure is simulated, we show that both types of disorder contribute to slow down water diffusion, and that allowing for protein motion, increasing the spatial dimensionality of the interface, reduces the anomalous character of hydration water. The rotation of water is, instead, altered by the energetic disorder only; indeed, when electrostatic interactions between the protein and water are switched off, water reorients even faster than in the bulk. The dynamics of water is also related to the collective structure--Ó voir the hydrogen bond (H-bond) network--formed by the solvent enclosing the protein surface. We show that, as expected for a full hydrated protein, when the protein surface offers pinning sites (charged or polar sites), the superficial water-water H-bond network percolates throughout the whole surface, hindering the water diffusion, whereas it does not when the protein surface lacks electrostatic interactions with water and the water diffusion is enhanced. PMID:17564431
Mixing and Transport in the Stockton Deep Water Ship Channel
NASA Astrophysics Data System (ADS)
Schmieder, P. J.; Ho, D. T.; Sharma, A. N.; Condon, M. E.; McDermott, J. A.; Clark, J. F.; Hurst, D. F.
The Stockton Deep Water Ship Channel (DWSC) section of the San Joaquin River suffers from hypoxia during the late summer and fall. Various factors have been hypothesized to contribute to low dissolved oxygen (DO) during this period, including thermal stratification, decreased freshwater water flow rates in the San Joaquin River, nutrient loading from municipal wastes and agricultural runoff, and geomorphic features of the dredged ship channel. To quantify the mixing and transport in the DWSC, an SF6 tracer release experiment was conducted in August 2005. On August 14, 2005, ca. 1.6 mol of SF6 was injected in the San Joaquin River, 13 km upstream from the DWSC. The tracer entered the DWSC within one day, and the tracer was mapped for 8 consecutive days following the injection. From the change in SF6 distribution with time, the longitudinal dispersion was determined to be 10.1 m2 s-1 and net advection was 1.2 km day-1 during the period of the study. Based on the decay of SF6 concentrations, the residence time for waters in the DWSC is ~3-4 days. This is considered a low estimate for the residence time as our survey was conducted under high flow conditions.
Corynebacterium diphtheriae: Identification and Characterization of a Channel-Forming Protein in the Cell Wall
Bettina Schiffler; Enrico Barth; Mamadou Daffe; Roland Benz
The cell wall fraction of the gram-positive, nontoxic Corynebacterium diphtheriae strain C8r() Tox ( ATCC 11913) contained a channel-forming protein, as judged from reconstitution experiments with artificial lipid bilayer experiments. The channel-forming protein was present in detergent-treated cell walls and in extracts of whole cells obtained using organic solvents. The protein had an apparent molecular mass of about 66 kDa
Punching Holes in Membranes: How Oligomeric Pore-Forming Proteins and Lipids Cooperate to Form Aqueous Channels in Membranes
NASA Astrophysics Data System (ADS)
Fradin, Cķcile; Satsoura, Dmitri; Andrews, David W.
Many important biological processes are carried out by a small number of proteins working together as a team to accomplish a specific task. Cooperation between the different proteins is often accomplished through the formation of a supramolecular complex, comprised of either identical or different subunits. Although the formation of protein assemblies is a favored mechanism throughout the cell, it becomes especially important in lipid membranes, as evidenced by the numerous cellular events that are either triggered by or result in the formation of protein complexes in membranes. However, due to the difficulties associated with the study of membrane proteins, the formation of oligomers in lipid membranes is perhaps one of the least understood cellular processes. In this chapter we focus our attention on a subset of membrane complexes Ś namely, those formed by proteins that are able to pass from a water-soluble to a transmembrane form in order to create a water-filled channel through the lipid membrane. These pore-forming proteins (PFPs) are found in many organisms throughout different kingdoms of life, from bacteria to human. They are often involved in cell death mechanisms through their capacity to break membrane permeability barriers, which can lead to dissipation of the membrane potential as well as introduction or leakage of enzymatic proteins. In fact, a large subset of the PFPs are toxins, and referred to in the literature as pore-forming toxins (PFTs). The association of several monomers into an oligomer is almost always an important aspect of the modus operandi of these proteins. Oligomerization can be useful in several ways: it results in structures large enough to delineate nanometer-size water-filled channels in lipid bilayers, it ensures the presence of large hydrophobic surfaces that can support insertion in the membrane, and it permits cooperative formation and insertion mechanisms.
Reaction rates and apparent toxicity of Houston Ship Channel waterĀ
Schneider, Peter William
was the Warburg Constant yolume Respirometer manometric method and the second technique was the conventional BOD dilution method using the standard BOD glass bottle. Tests were run on samples of water taken from the Houston Ship Channel. Tests showed... that the specific rate of reaction constant varies with both the depth and the location of the sample. Apparent toxic or inhibitory effects as defined in the thesis were also fo' nd to be present in many samples. The conditions prevailing , prior to the sampling...
Water uptake profile in a model ion-exchange membrane: conditions for water-rich channels.
Herbst, Daniel C; Witten, Thomas A; Tsai, Tsung-Han; Bryan Coughlin, E; Maes, Ashley M; Herring, Andrew M
Ionic conductivity in a polymeric fuel cell requires water uptake. Previous theoretical studies of water uptake used idealized parameters. We report a parameter-free prediction of the water-swelling behavior of a model fuel cell membrane. The model polymers, poly(methyl-butylene)-block-poly(vinylbenzyl-trimethylamine), form lamellar domains that absorb water in humid air. We use the Scheutjens-Fleer methodology to predict the resulting change in lamellar structure and compare with x-ray scattering. The results suggest locally uniform water distributions. However, under conditions where a PVBTMA and water mixture phase-separate, the two phases arrange into stripes with a dilute stripe sandwiched between two concentrated stripes. A small amount of water enhances conductivity most when it is partitioned into such channels, improving fuel-cell performance. PMID:25796265
Correlation of Apical Fluid-Regulating Channel Proteins with Lung Function in Human COPD Lungs
Zhao, Meimi; Liu, Shan-Lu; Huang, Yao; Idell, Steven; Li, Xiumin; Ji, Hong-Long
Links between epithelial ion channels and chronic obstructive pulmonary diseases (COPD) are emerging through animal model and in vitro studies. However, clinical correlations between fluid-regulating channel proteins and lung function in COPD remain to be elucidated. To quantitatively measure epithelial sodium channels (ENaC), cystic fibrosis transmembrane conductance regulator (CFTR), and aquaporin 5 (AQP5) proteins in human COPD lungs and to analyze the correlation with declining lung function, quantitative western blots were used. Spearman tests were performed to identify correlations between channel proteins and lung function. The expression of ? and ? ENaC subunits was augmented and inversely associated with lung function. In contrast, both total and alveolar type I (ATI) and II (ATII)-specific CFTR proteins were reduced. The expression level of CFTR proteins was associated with FEV1 positively. Abundance of AQP5 proteins and extracellular superoxide dismutase (SOD3) was decreased and correlated with spirometry test results and gas exchange positively. Furthermore, these channel proteins were significantly associated with severity of disease. Our study demonstrates that expression of ENaC, AQP5, and CFTR proteins in human COPD lungs is quantitatively associated with lung function and severity of COPD. These apically located fluid-regulating channels may thereby serve as biomarkers and potent druggable targets of COPD. PMID:25329998
Time-correlation analysis of simulated water motion in flexible and rigid gramicidin channels.
Chiu, S W; Jakobsson, E; Subramaniam, S; McCammon, J A
Molecular dynamics simulations have been done on a system consisting of the polypeptide membrane channel former gramicidin, plus water molecules in the channel and caps of waters at the two ends of the channel. In the absence of explicit simulation of the surrounding membrane, the helical form of the channel was maintained by artificial restraints on the peptide motion. The characteristic time constant of the artificial restraint was varied to assess the effect of the restraints on the channel structure and water motions. Time-correlation analysis was done on the motions of individual channel waters and on the motions of the center of mass of the channel waters. It is found that individual water molecules confined in the channel execute higher frequency motions than bulk water, for all degrees of channel peptide restraint. The center-of-mass motion of the chain of channel waters (which is the motion that is critical for transmembrane transport, due to the mandatory single filing of water in the channel) does not exhibit these higher frequency motions. The mobility of the water chain is dramatically reduced by holding the channel rigid. Thus permeation through the channel is not like flow through a rigid pipe; rather permeation is facilitated by peptide motion. For the looser restraints we used, the mobility of the water chain was not very much affected by the degree of restraint. Depending on which set of experiments is considered, the computed mobility of our water chain in the flexible channel is four to twenty times too high to account for the experimentally measured resistance of the gramicidin channel to water flow. From this result it appears likely that the peptide motions of an actual gramicidin channel embedded in a lipid membrane may be more restrained than in our flexible channel model, and that these restraints may be a significant modulator of channel permeability. For the completely rigid channel model the "trapping" of the water molecules in preferred positions throughout the molecular dynamics run precludes a reasonable assessment of mobility, but it seems to be quite low. PMID:1715766
Dramatic nano-fluidic properties of carbon nanotube membranes as a platform for protein channel mimetics
NASA Astrophysics Data System (ADS)
Hinds, Bruce
Carbon nanotubes have three key attributes that make them of great interest for novel membrane applications: 1) atomically flat graphite surface allows for ideal fluid slip boundary conditions and extremely fast flow rates 2) the cutting process to open CNTs inherently places functional chemistry at CNT core entrance for chemical selectivity and 3) CNT are electrically conductive allowing for electrochemical reactions and application of electric fields gradients at CNT tips. Pressure driven flux of a variety of solvents (H2O, hexane, decane ethanol, methanol) are 4-5 orders of magnitude higher than conventional Newtonian flow [Nature 2005, 438, 44] due to atomically flat graphite planes inducing nearly ideal slip conditions. However this is eliminated with selective chemical functionalization [ACS Nano 2011 5(5) 3867-3877] needed to give chemical selectivity. These unique properties allow us to explore the hypothesis of producing ``Gatekeeper'' membranes that mimic natural protein channels to actively pump through rapid nm-scale channels. With anionic tip functionality strong electroosmotic flow is induced by unimpeded cation flow with similar 10,000 fold enhancements [Nature Nano 2012 7(2) 133-39]. With enhanced power efficiency, carbon nanotube membranes were employed as the active element of a switchable transdermal drug delivery device that can facilitate more effective treatments of drug abuse and addiction. Recently methods to deposit Pt monolayers on CNT surface have been developed making for highly efficient catalytic platforms. Discussed are other applications of CNT protein channel mimetics, for large area robust engineering platforms, including water purification, flow battery energy storage, and biochemical/biomass separations. Carbon nanotubes have three key attributes that make them of great interest for novel membrane applications: 1) atomically flat graphite surface allows for ideal fluid slip boundary conditions and extremely fast flow rates 2) the cutting process to open CNTs inherently places functional chemistry at CNT core entrance for chemical selectivity and 3) CNT are electrically conductive allowing for electrochemical reactions and application of electric fields gradients at CNT tips. Pressure driven flux of a variety of solvents (H2O, hexane, decane ethanol, methanol) are 4-5 orders of magnitude higher than conventional Newtonian flow [Nature 2005, 438, 44] due to atomically flat graphite planes inducing nearly ideal slip conditions. However this is eliminated with selective chemical functionalization [ACS Nano 2011 5(5) 3867-3877] needed to give chemical selectivity. These unique properties allow us to explore the hypothesis of producing ``Gatekeeper'' membranes that mimic natural protein channels to actively pump through rapid nm-scale channels. With anionic tip functionality strong electroosmotic flow is induced by unimpeded cation flow with similar 10,000 fold enhancements [Nature Nano 2012 7(2) 133-39]. With enhanced power efficiency, carbon nanotube membranes were employed as the active element of a switchable transdermal drug delivery device that can facilitate more effective treatments of drug abuse and addiction. Recently methods to deposit Pt monolayers on CNT surface have been developed making for highly efficient catalytic platforms. Discussed are other applications of CNT protein channel mimetics, for large area robust engineering platforms, including water purification, flow battery energy storage, and biochemical/biomass separations. DOE EPSCoR (DE-FG02-07ER46375) and DARPA, W911NF-09-1-0267
Porin channels in Escherichia coli: studies with liposomes reconstituted from purified proteins.
Nikaido, H; Rosenberg, E Y
Rates of diffusion of uncharged and charged solute molecules through porin channels were determined by using liposomes reconstituted from egg phosphatidylcholine and purified Escherichia coli porins OmpF (protein 1a), OmpC (protein 1b), and PhoE (protein E). All three porin proteins appeared to produce channels of similar size, although the OmpF channel appeared to be 7 to 9% larger than the OmpC and PhoE channels in an equivalent radius. Hydrophobicity of the solute retarded the penetration through all three channels in a similar manner. The presence of one negative charge on the solute resulted in about a threefold reduction in penetration rates through OmpF and OmpC channels, whereas it produced two- to tenfold acceleration of diffusion through the PhoE channel. The addition of the second negatively charged group to the solutes decreased the diffusion rates through OmpF and OmpC channels further, whereas diffusion through the PhoE channel was not affected much. These results suggest that PhoE specializes in the uptake of negatively charged solutes. At the present level of resolution, no sign of true solute specificity was found in OmpF and OmpC channels; peptides, for example, diffused through both of these channels at rates expected from their molecular size, hydrophobicity, and charge. However, the OmpF porin channel allowed influx of more solute molecules per unit time than did the equivalent weight of the OmpC porin when the flux was driven by a concentration gradient of the same size. This apparent difference in "efficiency" became more pronounced with larger solutes, and it is likely to be the consequence of the difference in the sizes of OmpF and OmpC channels. PMID:6294049
Bioluminescence Methodology for the Detection of Protein¢Protein Interactions Within the Voltage-Gated Sodium Channel Macromolecular Complex
Shavkunov, Alexander; Panova, Neli; Prasai, Anesh; Veselenak, Ron; Bourne, Nigel; Stoilova-McPhie, Svetla
Abstract Protein¢protein interactions are critical molecular determinants of ion channel function and emerging targets for pharmacological interventions. Yet, current methodologies for the rapid detection of ion channel macromolecular complexes are still lacking. In this study we have adapted a split-luciferase complementation assay (LCA) for detecting the assembly of the voltage-gated Na+ (Nav) channel C-tail and the intracellular fibroblast growth factor 14 (FGF14), a functionally relevant component of the Nav channelosome that controls gating and targeting of Nav channels through direct interaction with the channel C-tail. In the LCA, two complementary N-terminus and C-terminus fragments of the firefly luciferase were fused, respectively, to a chimera of the CD4 transmembrane segment and the C-tail of Nav1.6 channel (CD4-Nav1.6-NLuc) or FGF14 (CLuc-FGF14). Co-expression of CLuc-FGF14 and CD4-Nav1.6-NLuc in live cells led to a robust assembly of the FGF14:Nav1.6 C-tail complex, which was attenuated by introducing single-point mutations at the predicted FGF14:Nav channel interface. To evaluate the dynamic regulation of the FGF14:Nav1.6 C-tail complex by signaling pathways, we investigated the effect of kinase inhibitors on the complex formation. Through a platform of counter screenings, we show that the p38/MAPK inhibitor, PD169316, and the I?B kinase inhibitor, BAY 11-7082, reduce the FGF14:Nav1.6 C-tail complementation, highlighting a potential role of the p38MAPK and the I?B/NF?B pathways in controlling neuronal excitability through protein¢protein interactions. We envision the methodology presented here as a new valuable tool to allow functional evaluations of protein¢channel complexes toward probe development and drug discovery targeting ion channels implicated in human disorders. PMID:22364545
Independently Gated Multiple Substates of an Epithelial Chloride-Channel Protein
NASA Astrophysics Data System (ADS)
Finn, Arthur L.; Dillard, Margaret; Gaido, Marcia
We have purified a protein from Necturus maculosus gallbladder cells that forms chloride channels in an artificial membrane. The same protein apparently can form channels that are highly selective for chloride but can have conductances varying from 9 to about 150 pS. The high-conductance channels are blocked by the monoclonal antibody used to purify the protein, but this antibody has no effect on the 9-pS channels. The observation that gating of the low- and high-conductance states is independent and that the antibody affects only the latter has implications regarding the control of chloride conductance in cell membranes and the different types of channels described in those cells.
Phosphoinositide control of membrane protein function: a frontier led by studies on ion channels.
Logothetis, Diomedes E; Petrou, Vasileios I; Zhang, Miao; Mahajan, Rahul; Meng, Xuan-Yu; Adney, Scott K; Cui, Meng; Baki, Lia
Anionic phospholipids are critical constituents of the inner leaflet of the plasma membrane, ensuring appropriate membrane topology of transmembrane proteins. Additionally, in eukaryotes, the negatively charged phosphoinositides serve as key signals not only through their hydrolysis products but also through direct control of transmembrane protein function. Direct phosphoinositide control of the activity of ion channels and transporters has been the most convincing case of the critical importance of phospholipid-protein interactions in the functional control of membrane proteins. Furthermore, second messengers, such as [Ca(2+)]i, or posttranslational modifications, such as phosphorylation, can directly or allosterically fine-tune phospholipid-protein interactions and modulate activity. Recent advances in structure determination of membrane proteins have allowed investigators to obtain complexes of ion channels with phosphoinositides and to use computational and experimental approaches to probe the dynamic mechanisms by which lipid-protein interactions control active and inactive protein states. PMID:25293526
Concentrating Toxoplasma gondii and Cyclospora cayetanensis from Surface Water and Drinking Water by Continuous Separation Channel Centrifugation
Aims: To evaluate the effectiveness of continuous separation channel centrifugation for concentrating Toxoplasma gondii and Cyclospora cayetanensis from drinking water and environmental waters. Methods and Results: Ready-to-seed vials with known quantities of Toxoplasma gondii a...
Molecular mechanism underlying ethanol activation of G-protein¢gated inwardly rectifying potassium channels
Bodhinathan, Karthik; Slesinger, Paul A.
Alcohol (ethanol) produces a wide range of pharmacological effects on the nervous system through its actions on ion channels. The molecular mechanism underlying ethanol modulation of ion channels is poorly understood. Here we used a unique method of alcohol-tagging to demonstrate that alcohol activation of a G-protein¢gated inwardly rectifying potassium (GIRK or Kir3) channel is mediated by a defined alcohol pocket through changes in affinity for the membrane phospholipid signaling molecule phosphatidylinositol 4,5-bisphosphate. Surprisingly, hydrophobicity and size, but not the canonical hydroxyl, were important determinants of alcohol-dependent activation. Altering levels of G protein G?? subunits, conversely, did not affect alcohol-dependent activation, suggesting a fundamental distinction between receptor and alcohol gating of GIRK channels. The chemical properties of the alcohol pocket revealed here might extend to other alcohol-sensitive proteins, revealing a unique protein microdomain for targeting alcohol-selective therapeutics in the treatment of alcoholism and addiction. PMID:24145411
Phycodnavirus potassium ion channel proteins question the virus molecular piracy hypothesis.
Hamacher, Kay; Greiner, Timo; Ogata, Hiroyuki; Van Etten, James L; Gebhardt, Manuela; Villarreal, Luis P; Cosentino, Cristian; Moroni, Anna; Thiel, Gerhard
Phycodnaviruses are large dsDNA, algal-infecting viruses that encode many genes with homologs in prokaryotes and eukaryotes. Among the viral gene products are the smallest proteins known to form functional K(+) channels. To determine if these viral K(+) channels are the product of molecular piracy from their hosts, we compared the sequences of the K(+) channel pore modules from seven phycodnaviruses to the K(+) channels from Chlorella variabilis and Ectocarpus siliculosus, whose genomes have recently been sequenced. C. variabilis is the host for two of the viruses PBCV-1 and NY-2A and E. siliculosus is the host for the virus EsV-1. Systematic phylogenetic analyses consistently indicate that the viral K(+) channels are not related to any lineage of the host channel homologs and that they are more closely related to each other than to their host homologs. A consensus sequence of the viral channels resembles a protein of unknown function from a proteobacterium. However, the bacterial protein lacks the consensus motif of all K(+) channels and it does not form a functional channel in yeast, suggesting that the viral channels did not come from a proteobacterium. Collectively, our results indicate that the viruses did not acquire their K(+) channel-encoding genes from their current algal hosts by gene transfer; thus alternative explanations are required. One possibility is that the viral genes arose from ancient organisms, which served as their hosts before the viruses developed their current host specificity. Alternatively the viral proteins could be the origin of K(+) channels in algae and perhaps even all cellular organisms. PMID:22685610
Phycodnavirus Potassium Ion Channel Proteins Question the Virus Molecular Piracy Hypothesis
Hamacher, Kay; Greiner, Timo; Ogata, Hiroyuki; Van Etten, James L.; Gebhardt, Manuela; Villarreal, Luis P.; Cosentino, Cristian; Moroni, Anna; Thiel, Gerhard
Phycodnaviruses are large dsDNA, algal-infecting viruses that encode many genes with homologs in prokaryotes and eukaryotes. Among the viral gene products are the smallest proteins known to form functional K+ channels. To determine if these viral K+ channels are the product of molecular piracy from their hosts, we compared the sequences of the K+ channel pore modules from seven phycodnaviruses to the K+ channels from Chlorella variabilis and Ectocarpus siliculosus, whose genomes have recently been sequenced. C. variabilis is the host for two of the viruses PBCV-1 and NY-2A and E. siliculosus is the host for the virus EsV-1. Systematic phylogenetic analyses consistently indicate that the viral K+ channels are not related to any lineage of the host channel homologs and that they are more closely related to each other than to their host homologs. A consensus sequence of the viral channels resembles a protein of unknown function from a proteobacterium. However, the bacterial protein lacks the consensus motif of all K+ channels and it does not form a functional channel in yeast, suggesting that the viral channels did not come from a proteobacterium. Collectively, our results indicate that the viruses did not acquire their K+ channel-encoding genes from their current algal hosts by gene transfer; thus alternative explanations are required. One possibility is that the viral genes arose from ancient organisms, which served as their hosts before the viruses developed their current host specificity. Alternatively the viral proteins could be the origin of K+ channels in algae and perhaps even all cellular organisms. PMID:22685610
Ogawa, Yasuhiro; Oses-Prieto, Juan; Kim, Moon Young; Horresh, Ido; Peles, Elior; Burlingame, Alma L.; Trimmer, James S.; Meijer, Dies; Rasband, Matthew N.
Clustered Kv1 K+ channels regulate neuronal excitability at juxtaparanodes of myelinated axons, axon initial segments (AIS), and cerebellar basket cell terminals (BCTs). These channels are part of a larger protein complex that includes cell adhesion molecules and scaffolding proteins. To identify proteins that regulate assembly, clustering, and/or maintenance of axonal Kv1 channel protein complexes, we immunoprecipitated Kv1.2 ? subunits, then used mass-spectrometry to identify interacting proteins. We found that ADAM22 (A Disintegrin And Metalloproteinase 22) is a component of the Kv1 channel complex, and that ADAM22 co-immunoprecipitates Kv1.2 and the MAGUKs PSD-93 and PSD-95. When co-expressed with MAGUKs in heterologous cells, ADAM22 and Kv1 channels are recruited into membrane surface clusters. However, co-expression of Kv1.2 with ADAM22 and MAGUKs does not alter channel properties. Among all the known Kv1 channel interacting proteins, only ADAM22 is found at every site where Kv1 channels are clustered. Analysis of Caspr-null mice showed that like other previously described juxtaparanodal proteins, disruption of the paranodal junction resulted in redistribution of ADAM22 into paranodal zones. Analysis of Caspr2-, PSD-93-, PSD-95-, and double PSD-93/PSD-95-null mice showed ADAM22 clustering at BCTs requires PSD-95, but ADAM22 clustering at juxtaparanodes requires neither PSD-93 nor PSD-95. In direct contrast, analysis of ADAM22-null mice demonstrated juxtaparanodal clustering of PSD-93 and PSD-95 requires ADAM22, whereas Kv1.2 and Caspr2 clustering is normal in ADAM22-null mice. Thus, ADAM22 is an axonal component of the Kv1 K+ channel complex that recruits MAGUKs to juxtaparanodes. PMID:20089912
The jellyfish green fluorescent protein: A new tool for studying ion channel expression and function
John Marshall; Raymond Molloy; Guy W. J Moss; James R Howe; Thomas E Hughes
Two methods are described for using the jellyfish green fluorescent protein (GFP) as a reporter gene for ion channel expression. GFP fluorescence can be used to identify the transfected cells, and to estimate the relative levels of ion channel expression, in cotransfection experiments. A GFP-NMDAR1 chimera can be constructed that produces a functional, fluorescent receptor subunit. These methods should facilitate
Optimum Dietary Protein to Energy Ratio for Channel Catfish Fingerlings, Ictalurus punctatus 1-4
Twenty-one semipurified diets were formulated to deter mine the optimum protein :energy ratio (P\\/E) for channel catfish finger- lings. Seven crude protein levels and three energy levels at each protein level were utilized. The protein, lipid, and digestible carbohydrate sources were hexane-extracted whole egg powder, salmon-corn oil mixture, and white dextrin, respectively. After an initial 2 week conditioning period, the
The influenza virus ion channel and maturation cofactor M2 is a cholesterol-binding protein
Cornelia Schroeder; Harald Heider; Elisabeth M÷ncke-Buchner; Tse-I Lin
The influenza-virus M2 protein has proton channel activity required for virus uncoating and maturation of hemagglutinin (HA) through low-pH compartments. The proton channel is cytotoxic in heterologous expression systems and can be blocked with rimantadine. In an independent, rimantadine-resistant function, M2, interacting with the M1 protein, controls the shape of virus particles. These bud from cholesterol-rich membrane rafts where viral
Ancient association between cation leak channels and Mid1 proteins is conserved in fungi and animals
Ghezzi, Alfredo; Liebeskind, Benjamin J.; Thompson, Ammon; Atkinson, Nigel S.; Zakon, Harold H.
Neuronal resting potential can tune the excitability of neural networks, affecting downstream behavior. Sodium leak channels (NALCN) play a key role in rhythmic behaviors by helping set, or subtly changing neuronal resting potential. The full complexity of these newly described channels is just beginning to be appreciated, however. NALCN channels can associate with numerous subunits in different tissues and can be activated by several different peptides and second messengers. We recently showed that NALCN channels are closely related to fungal calcium channels, which they functionally resemble. Here, we use this relationship to predict a family of NALCN-associated proteins in animals on the basis of homology with the yeast protein Mid1, the subunit of the yeast calcium channel. These proteins all share a cysteine-rich region that is necessary for Mid1 function in yeast. We validate this predicted association by showing that the Mid1 homolog in Drosophila, encoded by the CG33988 gene, is coordinately expressed with NALCN, and that knockdown of either protein creates identical phenotypes in several behaviors associated with NALCN function. The relationship between Mid1 and leak channels has therefore persisted over a billion years of evolution, despite drastic changes to both proteins and the organisms in which they exist. PMID:24639627
Channel-anchored protein kinase CK2 and protein phosphatase 1 reciprocally regulate KCNQ2-containing M-channels via phosphorylation of calmodulin.
Kang, Seungwoo; Xu, Mingxuan; Cooper, Edward C; Hoshi, Naoto
M-type potassium channels, encoded by the KCNQ family genes (KCNQ2-5), require calmodulin as an essential co-factor. Calmodulin bound to the KCNQ2 subunit regulates channel trafficking and stabilizes channel activity. We demonstrate that phosphorylation of calmodulin by protein kinase CK2 (casein kinase 2) rapidly and reversibly modulated KCNQ2 current. CK2-mediated phosphorylation of calmodulin strengthened its binding to KCNQ2 channel, caused resistance to phosphatidylinositol 4,5-bisphosphate depletion, and increased KCNQ2 current amplitude. Accordingly, application of CK2-selective inhibitors suppressed KCNQ2 current. This suppression was prevented by co-expression of CK2 phosphomimetic calmodulin mutants or pretreatment with a protein phosphatase inhibitor, calyculin A. We also demonstrated that functional CK2 and protein phosphatase 1 (PP1) were selectively tethered to the KCNQ2 subunit. We identified a functional KVXF consensus site for PP1 binding in the N-terminal tail of KCNQ2 subunit: mutation of this site augmented current density. CK2 inhibitor treatment suppressed M-current in rat superior cervical ganglion neurons, an effect negated by overexpression of phosphomimetic calmodulin or pretreatment with calyculin A Furthermore, CK2 inhibition diminished the medium after hyperpolarization by suppressing the M-current. These findings suggest that CK2-mediated phosphorylation of calmodulin regulates the M-current, which is tonically regulated by CK2 and PP1 anchored to the KCNQ2 channel complex. PMID:24627475
Channel Bow in Boiling Water Reactors - Hot Cell Examination Results and Correlation to Measured Bow
S. T. Mahmood; Y. P. Lin; M. A. Dubecky; K. Edsinger; E. V. Mader
An increase in frequency of fuel channel-control blade interference has been observed in Boiling Water Reactors (BWR) in recent years. Many of the channels leading to interference were found to bow towards the control blade in a manner that was inconsistent with the expected bow due to other effects. The pattern of bow appeared to indicate a new channel bow
Do cysteine residues regulate transient receptor potential canonical type 6 channel protein expression?
Thilo, Florian; Liu, Ying; Krueger, Katharina; F÷rste, Nora; Wittstock, Antje; Scholze, Alexandra; Tepel, Martin
The regulation of calcium influx through transient receptor potential canonical type 6 (TRPC6) channel is mandatory for the activity of human monocytes. We submit the first evidence that cysteine residues of homocysteine (HC) or acetylcysteine (ACC) affect TRPC6 expression in human monocytes. We observed that patients with chronic renal failure had significantly elevated HC levels and TRPC6 mRNA expression levels in monocytes compared with control subjects. We further observed that administration of HC or ACC significantly increased TRPC6 channel protein expression compared with control conditions. We, therefore, hypothesize that cysteine residues increase TRPC6 channel protein expression in humans. PMID:22004559
The ABC protein turned chloride channel whose failure causes cystic fibrosis
NASA Astrophysics Data System (ADS)
Gadsby, David C.; Vergani, Paola; Csanßdy, Lßszl¾
CFTR chloride channels are encoded by the gene mutated in patients with cystic fibrosis. These channels belong to the superfamily of ABC transporter ATPases. ATP-driven conformational changes, which in other ABC proteins fuel uphill substrate transport across cellular membranes, in CFTR open and close a gate to allow transmembrane flow of anions down their electrochemical gradient. New structural and biochemical information from prokaryotic ABC proteins and functional information from CFTR channels has led to a unifying mechanism explaining those ATP-driven conformational changes.
B. Jayaram; Tarun Jain
? Abstract Is it by design or by default that water molecules are observed at the interfaces of some protein-DNA complexes? Both experimental and theoretical studies on the thermodynamics of protein-DNA binding overwhelmingly support the extended hydrophobic view that water release from interfaces favors binding. Structural and en- ergy analyses indicate that the waters that remain at the interfaces of
Structure and Inhibition of the SARS Coronavirus Envelope Protein Ion Channel
Pervushin, Konstantin; Tan, Edward; Parthasarathy, Krupakar; Lin, Xin; Jiang, Feng Li; Yu, Dejie; Vararattanavech, Ardcharaporn; Soong, Tuck Wah; Liu, Ding Xiang; Torres, Jaume
The envelope (E) protein from coronaviruses is a small polypeptide that contains at least one ?-helical transmembrane domain. Absence, or inactivation, of E protein results in attenuated viruses, due to alterations in either virion morphology or tropism. Apart from its morphogenetic properties, protein E has been reported to have membrane permeabilizing activity. Further, the drug hexamethylene amiloride (HMA), but not amiloride, inhibited in vitro ion channel activity of some synthetic coronavirus E proteins, and also viral replication. We have previously shown for the coronavirus species responsible for severe acute respiratory syndrome (SARS-CoV) that the transmembrane domain of E protein (ETM) forms pentameric ?-helical bundles that are likely responsible for the observed channel activity. Herein, using solution NMR in dodecylphosphatidylcholine micelles and energy minimization, we have obtained a model of this channel which features regular ?-helices that form a pentameric left-handed parallel bundle. The drug HMA was found to bind inside the lumen of the channel, at both the C-terminal and the N-terminal openings, and, in contrast to amiloride, induced additional chemical shifts in ETM. Full length SARS-CoV E displayed channel activity when transiently expressed in human embryonic kidney 293 (HEK-293) cells in a whole-cell patch clamp set-up. This activity was significantly reduced by hexamethylene amiloride (HMA), but not by amiloride. The channel structure presented herein provides a possible rationale for inhibition, and a platform for future structure-based drug design of this potential pharmacological target. PMID:19593379
Membrane Protein Biosensor with Multi-Channel CMOS Impedance Extractor and Digitizer
Mason, Andrew
Membrane Protein Biosensor with Multi-Channel CMOS Impedance Extractor and Digitizer Chao Yang the development of biosensor arrays that harness the unique sensitivity and selectivity of membrane proteins frequency spectrum. This paper described the design and characterization of a biosensor array microsystem
Direct interaction between BKCa potassium channel and microtubule-associated protein 1A.
Park, Soo Mi; Liu, Guoxia; Kubal, Anup; Fury, Matthew; Cao, Luxiang; Marx, Steven O
The BKCa channel, a potassium channel that is allosterically activated by voltage and calcium, is expressed in both excitable and non-excitable cells. The channel plays an important role in regulating membrane excitability. The channel activity can be modulated by post-translational modifications such as phosphorylation. Recently, hippocampal BKCa channels were shown to be directly modulated by assembly/disassembly of the submembranous actin cytoskeleton. Here, we report that the BKCa channel physically interacts with the light chain of microtubule associated protein 1A (MAP1A). The light chain was isolated in a yeast two-hybrid screen of a human brain cDNA library. The specificity of the interaction was demonstrated in biochemical experiments utilizing GST fusion protein pulldown assays and reciprocal co-immunoprecipitations from rat brain. Furthermore, utilizing immunofluorescence, the BKCa channel and MAP1A co-localize in the Purkinje cell layer of the cerebellum. These studies identify a novel interaction between the C-terminal tail of the BKCa channel and the light chain of MAP1A, which enables channel association with and modulation by the cytoskeleton. PMID:15251455
Inhibition of G Protein-Activated Inwardly Rectifying K+ Channels by Phencyclidine
Kobayashi, Toru; Nishizawa, Daisuke; Ikeda, Kazutaka
Addictive drugs, such as opioids, ethanol, cocaine, amphetamine, and phencyclidine (PCP), affect many functions of the nervous system and peripheral organs, resulting in severe health problems. G protein-activated inwardly rectifying K+ (GIRK, Kir3) channels play an important role in regulating neuronal excitability through activation of various Gi/o protein-coupled receptors including opioid and CB1 cannabinoid receptors. Furthermore, the channels are directly activated by ethanol and inhibited by cocaine at toxic levels, but not affected by methylphenidate, methamphetamine, and 3,4-methylenedioxymethamphetamine (MDMA) at toxic levels. The primary pharmacological action of PCP is blockade of N-methyl-D-aspartate (NMDA) receptor channels that are associated with its psychotomimetic effects. PCP also interacts with several receptors and channels at relatively high concentrations. However, the molecular mechanisms underlying the various effects of PCP remain to be clarified. Here, we investigated the effects of PCP on GIRK channels using the Xenopus oocyte expression system. PCP weakly but significantly inhibited GIRK channels at micromolar concentrations, but not Kir1.1 and Kir2.1 channels. The PCP concentrations effective in inhibiting GIRK channels overlap clinically relevant brain concentrations in severe intoxication. The results suggest that partial inhibition of GIRK channels by PCP may contribute to some of the toxic effects after overdose. PMID:21886598
Emergence of adult aquatic insects was evaluated weekly with plexiglass traps positioned in two outdoor experimental channels from April through August 1977. One channel was seasonal Mississippi River water temperature and the other maintained at 10C above. Maximum water temperat...
Macroinvertebrates were sampled monthly from December 1976 to September 1977 in two 520 m long outdoor experimental channels. One channel was maintained at ambient seasonal Mississippi River water temperatures and the other 10C higher. Maximum water temperature reached 31.0C in t...
A process method and fixtures have been developed for fabricating square ; shaped Zircaloy-2 channels for the fuel assemblies used in the Vallecitos Boiling ; Water Reactor. Fourteen of these channels were made and assembled on fuel ; bundles. All of these bundles have been irradiated in the Vallecitos Boiling ; Water Reactor for various exposures and their suitability for
Specificity of cholesterol and analogs to modulate BK channels points to direct sterol-channel protein interactions.
Bukiya, Anna N; Belani, Jitendra D; Rychnovsky, Scott; Dopico, Alex M
The activity (Po) of large-conductance voltage/Ca(2+)-gated K(+) (BK) channels is blunted by cholesterol levels within the range found in natural membranes. We probed BK channel-forming ? (cbv1) subunits in phospholipid bilayers with cholesterol and related monohydroxysterols and performed computational dynamics to pinpoint the structural requirements for monohydroxysterols to reduce BK Po and obtain insights into cholesterol's mechanism of action. Cholesterol, cholestanol, and coprostanol reduced Po by shortening mean open and lengthening mean closed times, whereas epicholesterol, epicholestanol, epicoprostanol, and cholesterol trisnorcholenic acid were ineffective. Thus, channel inhibition by monohydroxysterols requires the ? configuration of the C3 hydroxyl and is favored by the hydrophobic nature of the side chain, while having lax requirements on the sterol A/B ring fusion. Destabilization of BK channel open state(s) has been previously interpreted as reflecting increased bilayer lateral stress by cholesterol. Lateral stress is controlled by the sterol molecular area and lipid monolayer lateral tension, the latter being related to the sterol ability to adopt a planar conformation in lipid media. However, we found that the differential efficacies of monohydroxysterols to reduce Po (cholesterol?coprostanol?cholestanol>epicholesterol) did not follow molecular area rank (coprostanol>epicholesterol>cholesterol>cholestanol). In addition, computationally predicted energies for cholesterol (effective BK inhibitor) and epicholesterol (ineffective) to adopt a planar conformation were similar. Finally, cholesterol and coprostanol reduced Po, yet these sterols have opposite effects on tight lipid packing and, likely, on lateral stress. Collectively, these findings suggest that an increase in bilayer lateral stress is unlikely to underlie the differential ability of cholesterol and related steroids to inhibit BK channels. Remarkably, ent-cholesterol (cholesterol mirror image) failed to reduce Po, indicating that cholesterol efficacy requires sterol stereospecific recognition by a protein surface. The BK channel phenotype resembled that of ? homotetramers. Thus, we hypothesize that a cholesterol-recognizing protein surface resides at the BK ? subunit itself. PMID:21149543
Natural channel protein inserts and functions in a completely artificial, solid-supported bilayer membrane
Zhang, Xiaoyan; Fu, Wangyang; Palivan, Cornelia G.; Meier, Wolfgang
Reconstitution of membrane proteins in artificial membrane systems creates a platform for exploring their potential for pharmacological or biotechnological applications. Previously, we demonstrated amphiphilic block copolymers as promising building blocks for artificial membranes with long-term stability and tailorable structural parameters. However, the insertion of membrane proteins has not previously been realized in a large-area, stable, and solid-supported artificial membrane. Here, we show the first, preliminary model of a channel membrane protein that is functionally incorporated in a completely artificial polymer, tethered, solid-supported bilayer membrane (TSSBM). Unprecedented ionic transport characteristics that differ from previous results on protein insertion into planar, free-standing membranes, are identified. Our findings mark a change in understanding protein insertion and ion flow within natural channel proteins when inserted in an artificial TSSBM, thus holding great potential for numerous applications such as drug screening, trace analyzing, and biosensing. PMID:23846807
Effects of C-reactive protein on K+ channel interaction protein 2 in cardiomyocytes
Xie, Yong; Mai, Jing-Ting; Wang, Fei; Lin, Yong-Qing; Yuan, Wo-Liang; Luo, Nian-Sang; Fang, Ming-Cheng; Wang, Jing-Feng; Chen, Yang-Xin
Several studies have found that C-reactive protein (CRP) was associated with QTc interval prolongation and ventricular arrhythmia. However, little is known about the mechanisms involved. K+ channel interaction protein 2 (KChIP2) is a necessary subunit for the formation of transient outward potassium current (Ito.f) which plays a critical role in early repolarization and QTc interval of heart. In this study, we aimed to evaluate the effects of CRP on KChIP2 and Ito.f in cardiomyocytes and to explore the potential mechanism. The neonatal mice ventricular cardiomyocytes were cultured and treated with CRP at different concentrations. The expression of KChIP2 was detected by real time quantitative PCR and Western blot. In addition, Ito.f current density was evaluated by whole cell patch clamp techniques. Our results showed that CRP significantly decreased the mRNA and protein expression of KChIP2 in time and doses dependent manners (P < 0.05), and also reduced the current density of Ito.f (P < 0.05). In addition, CRP increased the expression of NF-?B and decreased I?B? expression without significant influence on the expression of ERK1/2 and JNK. Meanwhile, the NF-?B inhibitor PDTC significantly attenuated the effects of CRP on KChIP2 and Ito.f current density. In conclusion, CRP could significantly down-regulate KChIP2 expression and reduce current density of Ito.f partly through NF-?B pathway, suggesting that CRP may directly or indirectly influence QTc interval and arrhythmia via influencing KChIP2 expression and Ito.f current density of cardiomyocytes. PMID:26175853
Channel wall coating on a poly-(methyl methacrylate) CE microchip by thermal immobilization of a cellulose derivative for size-based protein separation.
Okada, Hiroki; Kaji, Noritada; Tokeshi, Manabu; Baba, Yoshinobu
We demonstrate channel wall coating using a cellulose derivative on a poly-(methyl methacrylate) (PMMA) CE microchip to eliminate EOF disturbing protein separation. The channel walls were modified by preconditioning with a solution containing the cellulose derivative and then thermally evaporating the solution to produce hydrophilic channel walls which prevent adsorption of analytes via a hydrophobic interaction. When the PMMA substrate was coated with the cellulose derivative hydroxypropylmethylcellulose (HPMC) 90SH, the water contact angle on the coated substrate was decreased (up to 15 degrees ) and EOF was significantly suppressed (up to 4.0 x 10(-6) cm2.V(-1)s(-1)). Three proteins (20.5, 68.0, and 114.6 kDa) were successfully separated on the 0.15% HPMC 90SH-coated channel walls with good reproducibility of migration time (RSD <1.75%) and high efficiency (theoretical plate number per meter: 2.62 x 10(5)). PMID:18072224
Visualizing Water Molecules in Transmembrane Proteins Using Radiolytic Labeling Methods
Orban, T.; Gupta, S; Palczewski, K; Chance, M
Essential to cells and their organelles, water is both shuttled to where it is needed and trapped within cellular compartments and structures. Moreover, ordered waters within protein structures often colocalize with strategically placed polar or charged groups critical for protein function, yet it is unclear if these ordered water molecules provide structural stabilization, mediate conformational changes in signaling, neutralize charged residues, or carry out a combination of all these functions. Structures of many integral membrane proteins, including G protein-coupled receptors (GPCRs), reveal the presence of ordered water molecules that may act like prosthetic groups in a manner quite unlike bulk water. Identification of 'ordered' waters within a crystalline protein structure requires sufficient occupancy of water to enable its detection in the protein's X-ray diffraction pattern, and thus, the observed waters likely represent a subset of tightly bound functional waters. In this review, we highlight recent studies that suggest the structures of ordered waters within GPCRs are as conserved (and thus as important) as conserved side chains. In addition, methods of radiolysis, coupled to structural mass spectrometry (protein footprinting), reveal dynamic changes in water structure that mediate transmembrane signaling. The idea of water as a prosthetic group mediating chemical reaction dynamics is not new in fields such as catalysis. However, the concept of water as a mediator of conformational dynamics in signaling is just emerging, because of advances in both crystallographic structure determination and new methods of protein footprinting. Although oil and water do not mix, understanding the roles of water is essential to understanding the function of membrane proteins.
Signatures of protein structure in the cooperative gating of mechanosensitive ion channels
NASA Astrophysics Data System (ADS)
Kahraman, Osman; Klug, William S.; Haselwandter, Christoph A.
Membrane proteins deform the surrounding lipid bilayer, which can lead to membrane-mediated interactions between neighboring proteins. Using the mechanosensitive channel of large conductance (MscL) as a model system, we demonstrate how the observed differences in protein structure can affect membrane-mediated interactions and cooperativity among membrane proteins. We find that distinct oligomeric states of MscL lead to distinct gateway states for the clustering of MscL, and predict signatures of MscL structure and spatial organization in the cooperative gating of MscL. Our modeling approach establishes a quantitative relation between the observed shapes and the cooperative function of membrane proteins.
Functional reconstitution and channel activity measurements of purified wildtype and mutant CFTR protein.
Eckford, Paul D W; Li, Canhui; Bear, Christine E
The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a unique channel-forming member of the ATP Binding Cassette (ABC) superfamily of transporters. The phosphorylation and nucleotide dependent chloride channel activity of CFTR has been frequently studied in whole cell systems and as single channels in excised membrane patches. Many Cystic Fibrosis-causing mutations have been shown to alter this activity. While a small number of purification protocols have been published, a fast reconstitution method that retains channel activity and a suitable method for studying population channel activity in a purified system have been lacking. Here rapid methods are described for purification and functional reconstitution of the full-length CFTR protein into proteoliposomes of defined lipid composition that retains activity as a regulated halide channel. This reconstitution method together with a novel flux-based assay of channel activity is a suitable system for studying the population channel properties of wild type CFTR and the disease-causing mutants F508del- and G551D-CFTR. Specifically, the method has utility in studying the direct effects of phosphorylation, nucleotides and small molecules such as potentiators and inhibitors on CFTR channel activity. The methods are also amenable to the study of other membrane channels/transporters for anionic substrates. PMID:25867140
SDS capillary gel electrophoresis of proteins in microfabricated channels
Yao, Shao; Anex, Deon S.; Caldwell, W. Brett; Arnold, Don W.; Smith, Katherine B.; Schultz, Peter G.
Analysis of variations in the concentrations or structures of biomolecules (e.g., mRNAs, proteins, peptides, natural products) that occur either naturally or in response to environmental or genetic perturbations can provide important insight into complex biological processes. Many biological samples are mixtures that require a separation step before quantitation of variations in the individual components. Two-dimensional denaturing gel electrophoresis has been used very effectively to separate complex mixtures of proteins, but it is time consuming and requires considerable amounts of sample. Microchannel-based separations have proven very effective in rapidly separating small amounts of nucleic acids; more recently, isoelectric focusing of proteins also has been adapted to the microchannel format. Here, we describe microchannel-based SDS capillary gel electrophoresis of proteins and demonstrate the speed and high resolution it provides. This development is an important step toward the miniaturization and integration of multidimensional and array separation methods for complex protein mixtures. PMID:10318890
Structure and chromosomal localization of a human water channel (AQP3) gene
Ishibashi, Kenichi; Sasaki, Sei; Saito, Fumiko [Tokyo Medical and Dental Univ. (Japan)] [and others] [Tokyo Medical and Dental Univ. (Japan); and others
A cDNA encoding rat AQP3, a water channel and a member of the MIP family, that is expressed predominantly in kidney medulla and colon was cloned recently. To determine the structure, tissue distribution, and chromosomal localization of the human AQP3 gene, the authors screened a human kidney cDNA library with rat AQP3 probe and isolated a cDNA coding for human AQP3 protein. The deduced amino acid sequence of human AQP3 was 91% identical to rat AQP3. Human AQP3 mRNA was expressed in colon, kidney, liver, pancreas, lung, peripheral leukocytes, spleen, and prostate. The human AQP3 gene was mapped to 7q36.2-q36.3 by chromosome fluorescence in situ hybridization. 10 refs., 3 figs.
Arsenic removal from flowing irrigation water in bangladesh: impacts of channel properties.
Lineberger, Ethan M; Badruzzaman, A Borhan M; Ali, M Ashraf; Polizzotto, Matthew L
Across Bangladesh, dry-season irrigation with arsenic-contaminated well water is loading arsenic onto rice paddies, leading to increased arsenic concentrations in plants, diminished crop yields, and increased human health risks. As irrigation water flows through conveyance channels between wells and rice fields, arsenic concentrations change over space and time, indicating that channels may provide a location for removing arsenic from solution. However, few studies have systematically evaluated the processes controlling arsenic concentrations in irrigation channels, limiting the ability to manipulate these systems and enhance arsenic removal from solution. The central goal of this study was to quantify how channel design affected removal of dissolved arsenic from flowing irrigation water. Field experiments were conducted in Bangladesh using a chemically constant source of arsenic-contaminated irrigation water and an array of constructed channels with varying geometries. The resulting hydraulic conditions affected the quantity of arsenic removed from solution within the channels by promoting known hydrogeochemical processes. Channels three times the width of control channels removed ?3 times the mass of arsenic over 32 min of flowing conditions, whereas negligible arsenic removal was observed in tarp-lined channels, which prevented soil-water contact. Arsenic removal from solution was ?7 times higher in a winding, 200-m-long channel than in the straight, 45-m-long control channels. Arsenic concentrations were governed by oxidative iron-arsenic coprecipitation within the water column, sorption to soils, and phosphate competition. Collectively, these results suggest that better design and management of irrigation channels may play a part in arsenic mitigation strategies for rice fields in Southern Asia. PMID:25602413
Structure of the SecY channel during initiation of protein translocation
Park, Eunyong; Mķnķtret, Jean-Franńois; Gumbart, James C.; Ludtke, Steven J.; Li, Weikai; Whynot, Andrew
Many secretory proteins are targeted by signal sequences to a protein-conducting channel, formed by prokaryotic SecY- or eukaryotic Sec61-complexes, and are translocated across the membrane during their synthesis1,2. Crystal structures of the inactive channel show that the SecY subunit of the heterotrimeric complex consists of two halves that form an hourglass-shaped pore with a constriction in the middle of the membrane and a lateral gate that faces the lipid phase3-5. The closed channel has an empty cytoplasmic funnel and an extracellular funnel that is filled with a small helical domain, called the plug. During initiation of translocation, a ribosome¢nascent chain complex binds to the SecY/Sec61 complex, resulting in insertion of the nascent chain. However, the mechanism of channel opening during translocation is unclear. Here, we have addressed this question by determining structures of inactive and active ribosome¢channel complexes with cryo-electron microscopy. Non-translating ribosome¢SecY channel complexes derived from Methanococcus jannaschii or Escherichia coli show the channel in its closed state, and indicate that ribosome binding per se causes only minor changes. The structure of an active E. coli ribosome¢channel complex demonstrates that the nascent chain opens the channel, causing mostly rigid body movements of the N- and C-terminal halves of SecY. In this early translocation intermediate, the polypeptide inserts as a loop into the SecY channel with the hydrophobic signal sequence intercalated into the open lateral gate. The nascent chain also forms a loop on the cytoplasmic surface of SecY rather than directly entering the channel. PMID:24153188
Radiolytic mapping of solvent-contact surfaces in Photosystem II of higher plants: experimental identification of putative water channels within the photosystem.
Frankel, Laurie K; Sallans, Larry; Bellamy, Henry; Goettert, Jost S; Limbach, Patrick A; Bricker, Terry M
Photosystem II uses water as an enzymatic substrate. It has been hypothesized that this water is vectored to the active site for water oxidation via water channels that lead from the surface of the protein complex to the Mn4O5Ca metal cluster. The radiolysis of water by synchrotron radiation produces amino acid residue-modifying OH(Ģ) and is a powerful technique to identify regions of proteins that are in contact with water. In this study, we have used this technique to oxidatively modify buried amino acid residues in higher plant Photosystem II membranes. Fourier transform ion cyclotron resonance mass spectrometry was then used to identify these oxidized amino acid residues that were located in several core Photosystem II subunits (D1, D2, CP43, and CP47). While, as expected, the majority of the identified oxidized residues (?75%) are located on the solvent-exposed surface of the complex, a number of buried residues on these proteins were also modified. These residues form groups which appear to lead from the surface of the complex to the Mn4O5Ca cluster. These residues may be in contact with putative water channels in the photosystem. These results are discussed within the context of a number of largely computational studies that have identified putative water channels in Photosystem II. PMID:23814046
Effect of Reynolds number on 2-D protein crystallization at the air/water interface
NASA Astrophysics Data System (ADS)
Young, James; Posada, David; Hirsa, Amir; Lopez, Juan
X-ray diffraction is the primary technique used to obtain a detailed description of a protein on the molecular level and as such, has yielded essential information about protein structures and protein-ligand interactions. However a major drawback of this technique is that the protein must first be crystallized which is often a very difficult and inefficient process. It has been shown previously that the process of two-dimensional protein crystallization on lipid monolayers at the air/water interface can be enhanced by a shearing flow. Here we examine the relationship between Reynolds number and the crystal growth process using the deep-channel surface viscometer geometry, which consists of an annular region bounded by stationary inner and outer cylinders and driven by a constant rotation of the floor. The interfacial velocity measurements are compared to Navier-Stokes computations with the Boussinesq-Scriven surface model.
Bioinformatic Characterization of the Trimeric Intracellular Cation-Specific Channel Protein Family
Abe L. F. Silverio; Milton H. Saier
Trimeric intracellular cation-specific (TRIC) channels are integral to muscle excitation¢contraction coupling. TRIC channels\\u000a provide counter-ionic flux when calcium is rapidly transported from intracellular stores to the cell cytoplasm. Until recently,\\u000a knowledge of the presence of these proteins was limited to animals. We analyzed the TRIC family and identified a profusion\\u000a of prokaryotic family members with topologies and motifs similar to
ION CHANNELS, RECEPTORS AND TRANSPORTERS Determinants of the voltage dependence of G protein
Dolphin, Annette C.
in a previous study that, although G-mediated inhibitory modulation of CaV2.2 channels did not require constructs with CaV2.2 and 2-2, studied modula- tion by the activation of the dopamine D2 receptor, and alsoV subunits in the plasma membrane expression and G protein modulation of CaV2.2 calcium channels, by mutating
Theoretical models of the ion channel structure of amyloid beta-protein.
Durell, S R; Guy, H R; Arispe, N; Rojas, E; Pollard, H B
Theoretical methods are used to develop models for the ion channel structure of the membrane-bound amyloid beta-protein. This follows recent observations that the beta-protein forms cation-selective channels in lipid bilayers in vitro. Amyloid beta-protein is the main component of the extracellular plaques in the brain that are characteristic of Alzheimer's disease. Based on the amino acid sequence and the unique environment of the membrane, the secondary structure of the 40-residue beta-protein is predicted to form a beta-hairpin followed by a helix-turn-helix motif. The channel structures were-designed as aggregates of peptide subunits in identical conformations. Three types of models were developed that are distinguished by whether the pore is formed by the beta-hairpins, the middle helices, or by the more hydrophobic C-terminal helices. The latter two types can be converted back and forth by a simple conformational change, which would explain the variable conduction states observed for a single channel. It is also demonstrated how lipid headgroups could be incorporated into the pore lining, and thus affect the ion selectivity. The atomic-scale detail of the models make them useful for designing experiments to determine the real structure of the channel, and thus further the understanding of peptide channels in general. In addition, if beta-protein-induced channel activity is found to be the cause of cell death in Alzheimer's disease, then the models may be helpful in designing counteracting drugs. Images FIGURE 2 FIGURE 3 FIGURE 4 FIGURE 5 FIGURE 6 FIGURE 7 PMID:7535109
The glutamic acid-rich protein is a gating inhibitor of cyclic nucleotide-gated channels.
Michalakis, Stylianos; Zong, Xiangang; Becirovic, Elvir; Hammelmann, Verena; Wein, Thomas; Wanner, Klaus T; Biel, Martin
The cyclic nucleotide-gated (CNG) cation channel of rod photoreceptors is a heterotetramer consisting of homologous CNGA1 and CNGB1a subunits. While CNGA1 is indispensable for channel activation, the specific role of CNGB1a in this process has remained elusive. Here, we show that the N-terminal glutamic acid-rich protein (GARP) domain of CNGB1a and soluble GARP2, which corresponds to the proximal portion of the GARP domain, act as autoinhibitory domains that decrease the opening probability of the CNG channel. In the presence of mutations that structurally impair the cyclic nucleotide-binding domain (CNBD) of CNGB1a, the GARP domain completely abolishes channel activity. In agreement with an inhibitory function of GARP, the activity of mutant CNG channels could be fully restored by deletion of the GARP domain. We identified two sequences within the GARP domain that confer most of the inhibitory effect and demonstrate that the profound inhibition imposed by the GARP domain is caused by direct and autonomous protein-protein interaction with the CNG channel complex. In wild-type rod CNG channels, this inhibitory effect can be relieved by binding of cGMP to the CNBD of CNGB1a. In conclusion, we propose that the N terminus of CNGB1a and soluble GARPs act as molecular gate keepers that control the activation of heteromeric rod CNG channels. Our results suggest that the GARP domain has evolved in rod photoreceptors to reduce current noise resulting from openings of CNG channels in the absence of cGMP. PMID:21209198
Membrane Incorporation, Channel Formation, and Disruption of Calcium Homeostasis by Alzheimer's ?-Amyloid Protein
Kawahara, Masahiro; Ohtsuka, Isao; Yokoyama, Shoko; Kato-Negishi, Midori; Sadakane, Yutaka
Oligomerization, conformational changes, and the consequent neurodegeneration of Alzheimer's ?-amyloid protein (A?P) play crucial roles in the pathogenesis of Alzheimer's disease (AD). Mounting evidence suggests that oligomeric A?Ps cause the disruption of calcium homeostasis, eventually leading to neuronal death. We have demonstrated that oligomeric A?Ps directly incorporate into neuronal membranes, form cation-sensitive ion channels (ōamyloid channelsö), and cause the disruption of calcium homeostasis via the amyloid channels. Other disease-related amyloidogenic proteins, such as prion protein in prion diseases or ?-synuclein in dementia with Lewy bodies, exhibit similarities in the incorporation into membranes and the formation of calcium-permeable channels. Here, based on our experimental results and those of numerous other studies, we review the current understanding of the direct binding of A?P into membrane surfaces and the formation of calcium-permeable channels. The implication of composition of membrane lipids and the possible development of new drugs by influencing membrane properties and attenuating amyloid channels for the treatment and prevention of AD is also discussed. PMID:21547225
Pxmp2 Is a Channel-Forming Protein in Mammalian Peroxisomal Membrane
Rokka, Aare; Soininen, Raija; Immonen, Hanna L.; Pirilõ, Põivi L.; Bergmann, Ulrich; Sormunen, Raija T.; Weckstr÷m, Matti; Hiltunen, J. Kalervo
Background Peroxisomal metabolic machinery requires a continuous flow of organic and inorganic solutes across peroxisomal membrane. Concerning small solutes, the molecular nature of their traffic has remained an enigma. Methods/Principal Findings In this study, we show that disruption in mice of the Pxmp2 gene encoding Pxmp2, which belongs to a family of integral membrane proteins with unknown function, leads to partial restriction of peroxisomal membrane permeability to solutes in vitro and in vivo. Multiple-channel recording of liver peroxisomal preparations reveals that the channel-forming components with a conductance of 1.3 nS in 1.0 M KCl were lost in Pxmp2?/? mice. The channel-forming properties of Pxmp2 were confirmed with recombinant protein expressed in insect cells and with native Pxmp2 purified from mouse liver. The Pxmp2 channel, with an estimated diameter of 1.4 nm, shows weak cation selectivity and no voltage dependence. The long-lasting open states of the channel indicate its functional role as a protein forming a general diffusion pore in the membrane. Conclusions/Significance Pxmp2 is the first peroxisomal channel identified, and its existence leads to prediction that the mammalian peroxisomal membrane is permeable to small solutes while transfer of ōbulkyö metabolites, e.g., cofactors (NAD/H, NADP/H, and CoA) and ATP, requires specific transporters. PMID:19352492
Monte Carlo simulation of the water in a channel with charges.
Green, M E; Lewis, J
A Monte Carlo simulation of water in a channel with charges suggests the existence of water in immobile, high density, essentially glasslike form near the charges. The channel model has a conical section with an opening through which water molecules can pass, at the narrow end of the cone, and a cylindrical section at the other end. When the charges are placed near the narrow section of the model, the "glass" effectively blocks the channel; with the charges removed, the channel opens. The effect can be determined from the rate of passage of the water molecules through the pore, from the average orientation of the water molecule, and from distortion of the distribution of molecules. In the simulations carried out to date, no external ions have been considered. In addition to the energy, the Helmholtz free energy has been calculated. PMID:1706952
A MultiChannel Continuous Water Toxicity Monitoring System: Its Evaluation and Application to Water Discharged from a Power Plant
Byoung Chan Kim; Man Bock Gu
A multi-channel continuous water toxicity monitoring system was, after confirming the systems' performance, implemented to samples of water discharged from power plants to detect and classify their toxicity using several recombinant bioluminescent bacteria. Each channel of the system is composed of a series of two mini-bioreactors to enable a continuous operation, i.e., without system interruption due to highly toxic samples.
Water mobility, denaturation and the glass transition in proteins.
Porter, David; Vollrath, Fritz
A quantitative mechanism is presented that links protein denaturation and the protein-water glass transition through an energy criterion for the onset of mobility of strong protein-water bonds. Differences in the zero point vibrational energy in the ordered and disordered bonded states allow direct prediction of the two transition temperatures. While the onset of water mobility induces the same change in heat capacity for both transitions, the order-disorder transition of denaturation also predicts the observed excess enthalpy gain. The kinetics of the water and protein components through the glass transition are predicted and compared with dielectric spectroscopy observations. The energetic approach provides a consistent mechanism for processes such as refolding and aggregation of proteins involved in protein maintenance and adaptability, as the conformational constraints of strong water-amide bonds are lost with increased molecular mobility. Moreover, we suggest that the ordered state of peptide-water bonds is induced at the point of protein synthesis and could play a key role in the function of proteins through the enhancement of electronic activity by ferroelectric domains in the protein hydration shell, which is lost upon denaturation. PMID:22465032
The Small Hydrophobic Protein of the Human Respiratory Syncytial Virus Forms Pentameric Ion Channels*
Gan, Siok-Wan; Tan, Edward; Lin, Xin; Yu, Dejie; Wang, Juejin; Tan, Gregory Ming-Yeong; Vararattanavech, Ardcharaporn; Yeo, Chiew Ying; Soon, Cin Huang; Soong, Tuck Wah; Pervushin, Konstantin; Torres, Jaume
The small hydrophobic (SH) protein is encoded by the human respiratory syncytial virus. Its absence leads to viral attenuation in the context of whole organisms, and it prevents apoptosis in infected cells. Herein, we have examined the structure of SH protein in detergent micelles and in lipid bilayers, by solution NMR and attenuated total reflection-Fourier transform infrared spectroscopy, respectively. We found that SH protein has a single ?-helical transmembrane domain and forms homopentamers in several detergents. In detergent micelles, the transmembrane domain is flanked N-terminally by an ?-helix that forms a ring around the lumen of the pore and C-terminally by an extended ?-turn. SH protein was found in the plasma membrane of transiently expressing HEK 293 cells, which showed pH-dependent (acid-activated) channel activity. Channel activity was abolished in mutants lacking both native His residues, His22 and His51, but not when either His was present. Herein, we propose that the pentameric model of SH protein presented is a physiologically relevant conformation, albeit probably not the only one, in which SH contributes to RSV infection and replication. Viroporins are short (?100 amino acids) viral membrane proteins that form oligomers of a defined size, act as proton or ion channels, and in general enhance membrane permeability in the host. However, with some exceptions, their precise biological role of their channel activity is not understood. In general, viroporins resemble poorly specialized proteins but are nevertheless critical for viral fitness. In vivo, viruses lacking viroporins usually exhibit an attenuated or weakened phenotype, altered tropism, and diminished pathological effects. We have chosen to study the SH protein, 64 amino acids long, found in the human respiratory syncytial virus because of the effect of RSV on human health and the lack of adequate antivirals. We show that SH protein forms oligomers that behave as ion channels when activated at low pH. This study adds SH protein to a growing group of viroporins that have been structurally characterized. Although the precise biological role of this pentameric channel is still unknown, this report is nevertheless essential to fill some of the many gaps that exist in the understanding of SH protein function. PMID:22621926
Members of the chloride intracellular ion channel protein family demonstrate glutaredoxin-like enzymatic activity.
Al Khamici, Heba; Brown, Louise J; Hossain, Khondker R; Hudson, Amanda L; Sinclair-Burton, Alxcia A; Ng, Jane Phui Mun; Daniel, Elizabeth L; Hare, Joanna E; Cornell, Bruce A; Curmi, Paul M G; Davey, Mary W; Valenzuela, Stella M
The Chloride Intracellular Ion Channel (CLIC) family consists of six evolutionarily conserved proteins in humans. Members of this family are unusual, existing as both monomeric soluble proteins and as integral membrane proteins where they function as chloride selective ion channels, however no function has previously been assigned to their soluble form. Structural studies have shown that in the soluble form, CLIC proteins adopt a glutathione S-transferase (GST) fold, however, they have an active site with a conserved glutaredoxin monothiol motif, similar to the omega class GSTs. We demonstrate that CLIC proteins have glutaredoxin-like glutathione-dependent oxidoreductase enzymatic activity. CLICs 1, 2 and 4 demonstrate typical glutaredoxin-like activity using 2-hydroxyethyl disulfide as a substrate. Mutagenesis experiments identify cysteine 24 as the catalytic cysteine residue in CLIC1, which is consistent with its structure. CLIC1 was shown to reduce sodium selenite and dehydroascorbate in a glutathione-dependent manner. Previous electrophysiological studies have shown that the drugs IAA-94 and A9C specifically block CLIC channel activity. These same compounds inhibit CLIC1 oxidoreductase activity. This work for the first time assigns a functional activity to the soluble form of the CLIC proteins. Our results demonstrate that the soluble form of the CLIC proteins has an enzymatic activity that is distinct from the channel activity of their integral membrane form. This CLIC enzymatic activity may be important for protecting the intracellular environment against oxidation. It is also likely that this enzymatic activity regulates the CLIC ion channel function. PMID:25581026
Cooperative endocytosis of the endosomal SNARE protein syntaxin-8 and the potassium channel TASK-1
Renigunta, Vijay; Fischer, Thomas; Zuzarte, Marylou; Kling, Stefan; Zou, Xinle; Siebert, Kai; Limberg, Maren M.; Rinnķ, Susanne; Decher, Niels; Schlichth÷rl, G³nter; Daut, J³rgen
The endosomal SNARE protein syntaxin-8 interacts with the acid-sensitive potassium channel TASK-1. The functional relevance of this interaction was studied by heterologous expression of these proteins (and mutants thereof) in Xenopus oocytes and in mammalian cell lines. Coexpression of syntaxin-8 caused a fourfold reduction in TASK-1 current, a corresponding reduction in the expression of TASK-1 at the cell surface, and a marked increase in the rate of endocytosis of the channel. TASK-1 and syntaxin-8 colocalized in the early endosomal compartment, as indicated by the endosomal markers 2xFYVE and rab5. The stimulatory effect of the SNARE protein on the endocytosis of the channel was abolished when both an endocytosis signal in TASK-1 and an endocytosis signal in syntaxin-8 were mutated. A syntaxin-8 mutant that cannot assemble with other SNARE proteins had virtually the same effect as wild-type syntaxin-8. Total internal reflection fluorescence microscopy showed formation and endocytosis of vesicles containing fluorescence-tagged clathrin, TASK-1, and/or syntaxin-8. Our results suggest that the unassembled form of syntaxin-8 and the potassium channel TASK-1 are internalized via clathrin-mediated endocytosis in a cooperative manner. This implies that syntaxin-8 regulates the endocytosis of TASK-1. Our study supports the idea that endosomal SNARE proteins can have functions unrelated to membrane fusion. PMID:24743596
Aquaporin-4 water channels and synaptic plasticity in the hippocampus.
Scharfman, Helen E; Binder, Devin K
Aquaporin-4 (AQP4) is the major water channel expressed in the central nervous system (CNS) and is primarily expressed in glial cells. Many studies have shown that AQP4 regulates the response of the CNS to insults or injury, but far less is known about the potential for AQP4 to influence synaptic plasticity or behavior. Recent studies have examined long-term potentiation (LTP), long-term depression (LTD), and behavior in AQP4 knockout (KO) and wild-type mice to gain more insight into its potential role. The results showed a selective effect of AQP4 deletion on LTP of the Schaffer collateral pathway in hippocampus using an LTP induction protocol that simulates pyramidal cell firing during theta oscillations (theta-burst stimulation; TBS). However, LTP produced by a different induction protocol was unaffected. There was also a defect in LTD after low frequency stimulation (LFS) in AQP4 KO mice. Interestingly, some slices from AQP4 KO mice exhibited LTD after TBS instead of LTP, or LTP following LFS instead of LTD. These data suggest that AQP4 and astrocytes influence the polarity of long-term synaptic plasticity (potentiation or depression). These potentially powerful roles expand the influence of AQP4 and astrocytes beyond the original suggestions related to regulation of extracellular potassium and water balance. Remarkably, AQP4 KO mice did not show deficits in basal transmission, suggesting specificity for long-term synaptic plasticity. The mechanism appears to be related to neurotrophins and specifically brain-derived neurotrophic factor (BDNF) because pharmacological blockade of neurotrophin trk receptors or scavenging ligands such as BDNF restored plasticity. The in vitro studies predicted effects in vivo of AQP4 deletion because AQP4 KO mice performed worse using a task that requires memory for the location of objects (object placement). However, performance on other hippocampal-dependent tasks was spared. The results suggest an unanticipated and selective role of AQP4 in synaptic plasticity and spatial memory, and underscore the growing appreciation of the role of glial cells in functions typically attributed to neurons. Implications for epilepsy are discussed because of the previous evidence that AQP4 influences seizures, and the role of synaptic plasticity in epileptogenesis. PMID:23684954
Groundwater-surface water ecotones at the upstream part of confluences in former river channels
Gudrun Bornettel; Andrew R. G. Large
Two slow flowing former channels of the Rh¶ne River, which are supplied by nutrient-poor groundwater and subject to backflows of nutrient-rich water from the Rhone, were studied in order to demonstrate the occurrence of an ecotone located upstream to their confluences with the main channel. In one of the channels, the long-term stability of the boundary zone compared to the
Remaining life prediction of irradiated fuel channels for Boiling Water Reactor by ultrasonic testing
S. P. Srivastava; S. P. Pandarkar; T. G. Unni; R. L. Suthar; A. Ramu
The fuel channel forms an outer cover of the fuel assembly for the Boiling Water Reactor at Tarapur Atomic Power Station, (TAPS) Tarapur. Fuel channels are made of Zircaloy-4 material having square section of 113.5 ū 113.5mm, wall thickness of 1.5mm and length of 4029mm. The two major factors which affect fuel channel's performance are `Bulge' and `Bow', mainly attributed
Thermodynamics of all-or-none water channel closure in red cells
Teresa F. Moura; Robert I. Macey; David Y. Chien; Daniel Karan; Helena Santos
Summary The relation of osmotic to diffusional water permeability of human red blood cells was compared after treating the cells with different concentrations of PCMBS (p-chloromercuribenzene sulfonate). After subtracting the PCMBS-insensitive permeability (presumably the water permeability of the lipid bilayer) from each, the ratio of osmotic to diffusional permeability remains invariant (ś11) as more and more water channels are inhibited
Channels for water efflux and influx involved in volume regulation of murine spermatozoa
C Callies; T G Cooper; C H Yeung
The nature of the membrane channels mediating water transport in murine spermatozoa adjusting to anisotonic conditions was investigated. The volume of spermatozoa subjected to physiologically relevant hypotonic conditions either simultaneously, or after isotonic pre-incubation, with putative water transport inhibitors was monitored. Experiments in which quinine prevented osmolyte efflux, and thus regulatory volume decrease (RVD), revealed whether water influx or efflux
Submonolayer measurements of adsorbed proteins in microfluidic channels
David Henry; Keith Lenghaus; Kerry A. Wilson; Melissa Hirsch-Kuchma; Jerry Jenkins; Shankar Sundaram; James J. Hickman
Microelectromechanical systems (MEMS), and soon nanoelectromechanical systems (NEMS), are projected to have a major impact in systems for toxin detection, proteomics and biomedical applications. This chapter describes experimental systems that were developed in our labs, as well as those being developed elsewhere, that are being used to understand protein deposition at sub-monolayer coverages in these devices. Modeling tools, used in
The small envelope protein of porcine reproductive and respiratory syndrome virus possesses ion channel protein-like properties
Lee, Changhee [Department of Pathobiology, Ontario Veterinary College, University of Guelph, Guelph, Ontario, N1G 2W1 (Canada); Yoo, Dongwan [Department of Pathobiology, Ontario Veterinary College, University of Guelph, Guelph, Ontario, N1G 2W1 (Canada)]. E-mail: firstname.lastname@example.com
The small envelope (E) protein of porcine reproductive and respiratory syndrome virus (PRRSV) is a hydrophobic 73 amino acid protein encoded in the internal open reading frame (ORF) of the bicistronic mRNA2. As a first step towards understanding the biological role of E protein during PRRSV replication, E gene expression was blocked in a full-length infectious clone by mutating the ATG translational initiation to GTG, such that the full-length mutant genomic clone was unable to synthesize the E protein. DNA transfection of PRRSV-susceptible cells with the E gene knocked-out genomic clone showed the absence of virus infectivity. P129-{delta}E-transfected cells however produced virion particles in the culture supernatant, and these particles contained viral genomic RNA, demonstrating that the E protein is essential for PRRSV infection but dispensable for virion assembly. Electron microscopy suggests that the P129-{delta}E virions assembled in the absence of E had a similar appearance to the wild-type particles. Strand-specific RT-PCR demonstrated that the E protein-negative, non-infectious P129-{delta}E virus particles were able to enter cells but further steps of replication were interrupted. The entry of PRRSV has been suggested to be via receptor-mediated endocytosis, and lysomotropic basic compounds and known ion-channel blocking agents both inhibited PRRSV replication effectively during the uncoating process. The expression of E protein in Escherichia coli-mediated cell growth arrests and increased the membrane permeability. Cross-linking experiments in cells infected with PRRSV or transfected with E gene showed that the E protein was able to form homo-oligomers. Taken together, our data suggest that the PRRSV E protein is likely an ion-channel protein embedded in the viral envelope and facilitates uncoating of virus and release of the genome in the cytoplasm.
The M2 Proteins of Influenza A and B Viruses are Single-Pass Proton Channels
Yajun Tang; Padmavati Venkataraman; Jared Knopman; Robert A. Lamb; Lawrence H. Pinto
This chapter summarizes and evaluates the evidence that the M2 proteins of influenza A and B viruses possess intrinsic ion\\u000a channel activity that is essential to the life cycle of the virus. Both of these proteins are homotetramers with fewer than\\u000a 100 residues per subunit, an N-terminal ectodomain and a single transmembrane (TM) domain. There is little amino acid homology
GTP-binding proteins mediate transmitter inhibition of voltage-dependent calcium channels
George G. Holz; Stanley G. Rane; Kathleen Dunlap
The modulation of voltage-dependent calcium channels by hormones and neurotransmitters has important implications for the control of many Ca2+-dependent cellular functions including exocytosis and contractility1-7. We made use of electrophysiological techniques, including whole-cell patch-clamp recordings from dorsal root ganglion (DRG) neurones, to demonstrate a role for GTP-binding proteins (G-proteins) as signal transducers in the noradrenaline- and gamma-aminobutyric acid (GABA)-induced inhibition
Ricean Shadowed Statistical Characterization of Shallow Water Acoustic Channels for Wireless Communications
Ruiz-Vega, F; Otero, P; Paris, J F
In this letter, the statistical behaviour of the shallow water acoustic channel for wireless communications is shown to be well characterized by the Ricean shadowed distribution, which has never been proposed for communication purposes on this type of channel. This characterization is clearly motivated from statistical and physical perspectives and has both theoretical and practical advantages compared to previously proposed models.
Using normal mode channel structure for narrow band underwater communications in shallow water
A. G. Silva; S. M. Jesus
Multipath and high temporal and spatial variability of the propagation environment causes severe signal degradation in shallow water acoustic digital communications. Among the many solutions that have been proposed the most known is adaptive equalisation where cyclic training signals are used to adapt the equaliser to the variability of the acoustic channel. When the channel is rapidly changing, equaliser coefficients
How water contributes to pressure and cold denaturation of proteins
Bianco, Valentino
The mechanisms of cold- and pressure-denaturation of proteins are matter of debate and are commonly understood as due to water-mediated interactions. Here we study several cases of proteins, with or without a unique native state, with or without hydrophilic residues, by means of a coarse-grain protein model in explicit solvent. We show, using Monte Carlo simulations, that taking into account how water at the protein interface changes its hydrogen bond properties and its density fluctuations is enough to predict protein stability regions with elliptic shapes in the temperature-pressure plane, consistent with previous theories. Our results clearly identify the different mechanisms with which water participates to denaturation and open the perspective to develop advanced computational design tools for protein engineering.
How water contributes to pressure and cold denaturation of proteins
Valentino Bianco; Giancarlo Franzese
The mechanisms of cold- and pressure-denaturation of proteins are matter of debate and are commonly understood as due to water-mediated interactions. Here we study several cases of proteins, with or without a unique native state, with or without hydrophilic residues, by means of a coarse-grain protein model in explicit solvent. We show, using Monte Carlo simulations, that taking into account how water at the protein interface changes its hydrogen bond properties and its density fluctuations is enough to predict protein stability regions with elliptic shapes in the temperature-pressure plane, consistent with previous theories. Our results clearly identify the different mechanisms with which water participates to denaturation and open the perspective to develop advanced computational design tools for protein engineering.
Building the water edge : a public event for art and artists at Fort Point Channel
Godwin, Audrey
The thesis deals with building the water edge at Fort Point Channel, between Congress Street and Summer Street Bridges. It serves as a public event that intends to establish continuity of movement along the waterfront. The ...
G protein-coupled receptor signaling to Kir channels in Xenopus oocytes.
Hatcher-Solis, Candice; Fribourg, Miguel; Spyridaki, Katerina; Younkin, Jason; Ellaithy, Amr; Xiang, Guoqing; Liapakis, George; Gonzalez-Maeso, Javier; Zhang, Hailin; Cui, Meng; Logothetis, Diomedes E
Kir3 (or GIRK) channels have been known for nearly three decades to be activated by direct interactions with the ?? subunits of heterotrimeric G (G???) proteins in a membrane-delimited manner. G? also interacts with GIRK channels and since PTX-sensitive G? subunits show higher affinity of interaction they confer signaling specificity to G Protein- Coupled Receptors (GPCRs) that normally couple to these G protein subunits. In heterologous systems, overexpression of non PTX-sensitive G? subunits scavenges the available G?? and biases GIRK activation through GPCRs that couple to these G? subunits. Moreover, all Kir channels rely on their direct interactions with the phospholipid PIP2 to maintain their activity. Thus, signals that activate phospholipase C (e.g. through Gq signaling) to hydrolyze PIP2 result in inhibition of Kir channel activity. In this review, we illustrate with experiments performed in Xenopus oocytes that Kir channels can be used efficiently as reporters of GPCR function through Gi, Gs or Gq signaling. The membrane-delimited nature of this expression system makes it highly efficient for constructing dose-response curves yielding highly reproducible apparent affinities of different ligands for each GPCR tested. PMID:25374032
Clapham, David E.
was purified from mouse testis that contained HSP70-2, a testis-specific chaperone, and CatSper, a novelSper was restricted to testis and localized to the principal piece of the sperm tail. CatSper protein is absent in Cat). CatSpers1-4 are expressed in testis and localized primarily to the principal piece of sperm tail (8
LEA proteins during water stress: not just for plants anymore.
Hand, Steven C; Menze, Michael A; Toner, Mehmet; Boswell, Leaf; Moore, Daniel
Late embryogenesis abundant (LEA) proteins are extremely hydrophilic proteins that were first identified in land plants. Intracellular accumulation is tightly correlated with acquisition of desiccation tolerance, and data support their capacity to stabilize other proteins and membranes during drying, especially in the presence of sugars like trehalose. Exciting reports now show that LEA proteins are not restricted to plants; multiple forms are expressed in desiccation-tolerant animals from at least four phyla. We evaluate here the expression, subcellular localization, biochemical properties, and potential functions of LEA proteins in animal species during water stress. LEA proteins are intrinsically unstructured in aqueous solution, but surprisingly, many assume their native conformation during drying. They are targeted to multiple cellular locations, including mitochondria, and evidence supports that LEA proteins stabilize vitrified sugar glasses thought to be important in the dried state. More in vivo experimentation will be necessary to fully unravel the multiple functional properties of these macromolecules during water stress. PMID:21034219
Intra-membrane molecular interactions of K%2B channel proteins : application to problems in biodefense and bioenergy.
Moczydlowski, Edward G.
Ion channel proteins regulate complex patterns of cellular electrical activity and ionic signaling. Certain K+ channels play an important role in immunological biodefense mechanisms of adaptive and innate immunity. Most ion channel proteins are oligomeric complexes with the conductive pore located at the central subunit interface. The long-term activity of many K+ channel proteins is dependent on the concentration of extracellular K+; however, the mechanism is unclear. Thus, this project focused on mechanisms underlying structural stability of tetrameric K+ channels. Using KcsA of Streptomyces lividans as a model K+ channel of known structure, the molecular basis of tetramer stability was investigated by: 1. Bioinformatic analysis of the tetramer interface. 2. Effect of two local anesthetics (lidocaine, tetracaine) on tetramer stability. 3. Molecular simulation of drug docking to the ion conduction pore. The results provide new insights regarding the structural stability of K+ channels and its possible role in cell physiology.
Crystallization of the large membrane protein complex photosystem I in a microfluidic channel.
Abdallah, Bahige G; Kupitz, Christopher; Fromme, Petra; Ros, Alexandra
Traditional macroscale protein crystallization is accomplished nontrivially by exploring a range of protein concentrations and buffers in solution until a suitable combination is attained. This methodology is time-consuming and resource-intensive, hindering protein structure determination. Even more difficulties arise when crystallizing large membrane protein complexes such as photosystem I (PSI) due to their large unit cells dominated by solvent and complex characteristics that call for even stricter buffer requirements. Structure determination techniques tailored for these "difficult to crystallize" proteins such as femtosecond nanocrystallography are being developed yet still need specific crystal characteristics. Here, we demonstrate a simple and robust method to screen protein crystallization conditions at low ionic strength in a microfluidic device. This is realized in one microfluidic experiment using low sample amounts, unlike traditional methods where each solution condition is set up separately. Second harmonic generation microscopy via second-order nonlinear imaging of chiral crystals (SONICC) was applied for the detection of nanometer- and micrometer-sized PSI crystals within microchannels. To develop a crystallization phase diagram, crystals imaged with SONICC at specific channel locations were correlated to protein and salt concentrations determined by numerical simulations of the time-dependent diffusion process along the channel. Our method demonstrated that a portion of the PSI crystallization phase diagram could be reconstructed in excellent agreement with crystallization conditions determined by traditional methods. We postulate that this approach could be utilized to efficiently study and optimize crystallization conditions for a wide range of proteins that are poorly understood to date. PMID:24191698
Crystallization of the Large Membrane Protein Complex Photosystem I in a Microfluidic Channel
Abdallah, Bahige G.; Kupitz, Christopher; Fromme, Petra; Ros, Alexandra
Traditional macroscale protein crystallization is accomplished non-trivially by exploring a range of protein concentrations and buffers in solution until a suitable combination is attained. This methodology is time consuming and resource intensive, hindering protein structure determination. Even more difficulties arise when crystallizing large membrane protein complexes such as photosystem I (PSI) due to their large unit cells dominated by solvent and complex characteristics that call for even stricter buffer requirements. Structure determination techniques tailored for these ædifficult to crystallizeÆ proteins such as femtosecond nanocrystallography are being developed, yet still need specific crystal characteristics. Here, we demonstrate a simple and robust method to screen protein crystallization conditions at low ionic strength in a microfluidic device. This is realized in one microfluidic experiment using low sample amounts, unlike traditional methods where each solution condition is set up separately. Second harmonic generation microscopy via Second Order Nonlinear Imaging of Chiral Crystals (SONICC) was applied for the detection of nanometer and micrometer sized PSI crystals within microchannels. To develop a crystallization phase diagram, crystals imaged with SONICC at specific channel locations were correlated to protein and salt concentrations determined by numerical simulations of the time-dependent diffusion process along the channel. Our method demonstrated that a portion of the PSI crystallization phase diagram could be reconstructed in excellent agreement with crystallization conditions determined by traditional methods. We postulate that this approach could be utilized to efficiently study and optimize crystallization conditions for a wide range of proteins that are poorly understood to date. PMID:24191698
G-Protein Inhibition of N- and P\\/Q-Type Calcium Channels: Distinctive Elementary Mechanisms and Their Functional Impact
Henry M. Colecraft; David L. Brody; David T. Yue
Voltage-dependent G-protein inhibition of presynaptic Ca 21 channels is a key mechanism for regulating synaptic efficacy. G-protein bg subunits produce such inhibition by binding to and shifting channel opening patterns from high to low open probability regimes, known respectively as \\
KCNQ1 channel modulation by KCNE proteins via the voltage-sensing domain.
Nakajo, Koichi; Kubo, Yoshihiro
The gating of the KCNQ1 potassium channel is drastically regulated by auxiliary subunit KCNE proteins. KCNE1, for example, slows the activation kinetics of KCNQ1 by two orders of magnitude. Like other voltage-gated ion channels, the opening of KCNQ1 is regulated by the voltage-sensing domain (VSD; S1-S4 segments). Although it has been known that KCNE proteins interact with KCNQ1 via the pore domain, some recent reports suggest that the VSD movement may be altered by KCNE. The altered VSD movement of KCNQ1 by KCNE proteins has been examined by site-directed mutagenesis, the scanning cysteine accessibility method (SCAM), voltage clamp fluorometry (VCF) and gating charge measurements. These accumulated data support the idea that KCNE proteins interact with the VSDs of KCNQ1 and modulate the gating of the KCNQ1 channel. In this review, we will summarize recent findings and current views of the KCNQ1 modulation by KCNE via the VSD. In this context, we discuss our recent findings that KCNE1 may alter physical interactions between the S4 segment (VSD) and the S5 segment (pore domain) of KCNQ1. Based on these findings from ourselves and others, we propose a hypothetical mechanism for how KCNE1 binding alters the VSD movement and the gating of the channel. PMID:25603957
Technology Transfer Automated Retrieval System (TEKTRAN)
A 2-year continuous production trial was conducted in earthen ponds to evaluate lysine supplementation of practical diets as a means to reduce the need for total dietary protein and limit nitrogenous waste production of channel catfish. Experimental diets consisted of three practical diets containi...
Protein electrostriction: a possibility of elastic deformation of the ? -hemolysin channel by the applied field
Oleg V. Krasilnikov; Petr G. Merzlyak; Liliya N. Yuldasheva; Maria F. Capistrano
While conformational flexibility of proteins is widely recognized as one of their functionally crucial features and enjoys proper attention for this reason, their elastic properties are rarely discussed. In ion channel studies, where the voltage-induced or ligand-induced conformational transitions, gating, are the leading topic of research, the elastic structural deformation by the applied electric field has never been addressed at
Towards mimicking natural protein channels with aligned carbon nanotube membranes for active drug delivery
Majumder, Mainak; Stinchcomb, Audra; Hinds, Bruce J.
Aims Carbon nanotube (CNT) membranes offer an exciting opportunity to mimic natural protein channels due to 1) a mechanism of dramatically enhanced fluid flow 2) ability to place ægatekeeperÆ chemistry at the entrance to pores 3) the ability for biochemical reactions to occur on gatekeeper molecules and 4) an ability to chemically functionalize each side of the membrane independently. Main methods Aligned CNT membranes were fabricated and CNT pore entrances modified with gatekeeper chemistry. Pressure driven fluid flow and diffusion experiments were performed to study the mechanisms of transport through CNTs. Key findings The transport mechanism through CNT membranes is primarily 1) ionic diffusion near bulk expectation 2) gas flow enhanced 1¢2 orders of magnitude primarily due to specular reflection 3) fluid flow 4¢5 orders of magnitude faster than conventional materials due to a nearly ideal slip-boundary interface. The transport can be modulated by ægatekeeperÆ chemistry at the pore entrance using steric hindrance, electrostatic attraction/repulsion, or biochemical state. The conformation of charged tethered molecules can be modulated by applied bias setting the stage for programmable drug release devices. Significance The membrane structure is mechanically far more robust than lipid bilayer films, allowing for large-scale chemical separations, delivery or sensing based on the principles of protein channels. The performance of protein channels is several orders of magnitude faster than conventional membrane materials. The fundamental requirements of mimicking protein channels are present in the CNT membrane system. PMID:19383500
Technology Transfer Automated Retrieval System (TEKTRAN)
Significant effort has been made to utilize alternative protein sources like cottonseed meal (CSM) and soybean meal (SBM) to replace fishmeal in channel catfish (Ictalurus punctatus) feed. These sources are readily available and have high nutritional value, but contain anti-nutritional factors (ANF...
Identification and characterisation of a functional aquaporin water channel (Anomala cuprea DRIP) in a coleopteran insect.
Nagae, Tomone; Miyake, Seiji; Kosaki, Shiho; Azuma, Masaaki
Water transport across the plasma membrane depends on the presence of the water channel aquaporin (AQP), which mediates the bulk movement of water through osmotic and pressure gradients. In terrestrial insects, which are solid and/or plant feeders, the entrance and exit of water is primarily executed along the alimentary tract, where the hindgut, particularly the rectum, is the major site of water conservation. A cDNA encoding the homologue of the water-specific Drosophila AQP [Drosophila integral protein (DRIP)] was identified through the RT-PCR of RNA isolated from the rectum of the cupreous chafer larvae, Anomala cuprea, a humus and plant root feeder. This gene (Anocu AQP1) has a predicted molecular mass of 26.471 kDa, similar to the DRIP clade of insect AQPs characterised from caterpillars, flies and several liquid-feeding insects. When expressed in Xenopus laevis oocytes, Anocu AQP1 showed the hallmarks of aquaporin-mediated water transport but no glycerol or urea permeability, and the reversible inhibition of elevated water transport through 1 mmol l(-1) HgCl2. This is the first experimental demonstration of the presence of a water-specific AQP, namely DRIP, in the Coleoptera. The genome of the model beetle Tribolium castaneum contains six putative AQP sequences, one of which (Trica-1a, XP_972862) showed the highest similarity to Anocu AQP1 (~60% amino acid identity). Anocu AQP1 is predominantly expressed in the rectum. Using a specific antibody raised against DRIP in the silkworm Bombyx mori (AQP-Bom1), Anocu AQP1 was localised to the apical plasma membrane of rectal epithelial cells, and lacking in the midgut and gastric caecal epithelia. Based on the BeetleBase prediction, there are three putative AQPs (Trica-3a, 3b, 3c: XP_970728, 970912, 970791) that are homologous to B. mori aquaglyceroporin [AQP-Bom2 (GLP)]. The immunocytochemical studies using the specific anti-peptide antibody against AQP-Bom2 revealed the presence of the GLP homologue at the apical plasma membrane of enterocytes in the midgut and gastric caeca. Thus, DRIP (Anocu AQP1) and the putative GLP share epithelial fluid-transporting roles along the alimentary tract in cupreous chafer larvae. PMID:23531819
Water dynamics clue to key residues in protein folding
Gao, Meng [State Key Laboratory for Turbulence and Complex Systems, and Department of Biomedical Engineering, and Center for Theoretical Biology, and Center for Protein Science, Peking University, Beijing 100871 (China)] [State Key Laboratory for Turbulence and Complex Systems, and Department of Biomedical Engineering, and Center for Theoretical Biology, and Center for Protein Science, Peking University, Beijing 100871 (China); Zhu, Huaiqiu, E-mail: email@example.com [State Key Laboratory for Turbulence and Complex Systems, and Department of Biomedical Engineering, and Center for Theoretical Biology, and Center for Protein Science, Peking University, Beijing 100871 (China)] [State Key Laboratory for Turbulence and Complex Systems, and Department of Biomedical Engineering, and Center for Theoretical Biology, and Center for Protein Science, Peking University, Beijing 100871 (China); Yao, Xin-Qiu [State Key Laboratory for Turbulence and Complex Systems, and Department of Biomedical Engineering, and Center for Theoretical Biology, and Center for Protein Science, Peking University, Beijing 100871 (China) [State Key Laboratory for Turbulence and Complex Systems, and Department of Biomedical Engineering, and Center for Theoretical Biology, and Center for Protein Science, Peking University, Beijing 100871 (China); Department of Biophysics, Kyoto University, Sakyo Kyoto 606-8502 (Japan); She, Zhen-Su, E-mail: email@example.com [State Key Laboratory for Turbulence and Complex Systems, and Department of Biomedical Engineering, and Center for Theoretical Biology, and Center for Protein Science, Peking University, Beijing 100871 (China)] [State Key Laboratory for Turbulence and Complex Systems, and Department of Biomedical Engineering, and Center for Theoretical Biology, and Center for Protein Science, Peking University, Beijing 100871 (China)
A computational method independent of experimental protein structure information is proposed to recognize key residues in protein folding, from the study of hydration water dynamics. Based on all-atom molecular dynamics simulation, two key residues are recognized with distinct water dynamical behavior in a folding process of the Trp-cage protein. The identified key residues are shown to play an essential role in both 3D structure and hydrophobic-induced collapse. With observations on hydration water dynamics around key residues, a dynamical pathway of folding can be interpreted.
A virus-encoded potassium ion channel is a structural protein in the chlorovirus Paramecium bursaria chlorella virus 1 virion
Romani, Giulia; Piotrowski, Adrianna; Hillmer, Stefan; Gurnon, James; Van Etten, James L.; Moroni, Anna; Thiel, Gerhard
Most chloroviruses encode small K+ channels, which are functional in electrophysiological assays. The experimental finding that initial steps in viral infection exhibit the same sensitivity to channel inhibitors as the viral K+ channels has led to the hypothesis that the channels are structural proteins located in the internal membrane of the virus particles. This hypothesis was questioned recently because proteomic studies failed to detect the channel protein in virions of the prototype chlorovirus Paramecium bursaria chlorella virus 1 (PBCV-1). Here, we used a mAb raised against the functional K+ channel from chlorovirus MA-1D to search for the viral K+ channel in the virus particle. The results showed that the antibody was specific and bound to the tetrameric channel on the extracellular side. The antibody reacted in a virus-specific manner with protein extracts from chloroviruses that encoded channels similar to that from MA-1D. There was no cross-reactivity with chloroviruses that encoded more diverse channels or with a chlorovirus that lacked a K+ channel gene. Together with electron microscopic imaging, which revealed labelling of individual virus particles with the channel antibody, these results establish that the viral particles contain an active K+ channel, presumably located in the lipid membrane that surrounds the DNA in the mature virions. PMID:23918407
Vibrational energy flow through the green fluorescent protein-water interface: communication maps and thermal boundary conductance.
Xu, Yao; Leitner, David M
We calculate communication maps for green fluorescent protein (GFP) to elucidate energy transfer pathways between the chromophore and other parts of the protein in the ground and excited state. The approach locates energy transport channels from the chromophore to remote regions of the protein via residues and water molecules that hydrogen bond to the chromophore. We calculate the thermal boundary conductance between GFP and water over a wide range of temperature and find that the interface between the protein and the cluster of water molecules in the ?-barrel poses negligible resistance to thermal flow, consistent with facile vibrational energy transfer from the chromophore to the ?-barrel waters observed in the communication maps. PMID:24471982
Microfiltration: Effect of channel diameter on limiting flux and serum protein removal.
Hurt, E E; Adams, M C; Barbano, D M
Our objective was to determine the limiting flux and serum protein (SP) removal at 8, 9 and 10% true protein (TP) in the retentate recirculation loop using 0.1-Ąm ceramic graded permeability (GP) microfiltration (MF) membranes with 3mm channel diameters (CD). An additional objective was to compare the limiting flux and SP removal between 0.1-Ąm ceramic GP membranes with 3mm CD and previous research using 4-mm CD membranes. The MF system was operated at 50░C, using a diluted milk protein concentrate with 85% protein on a total solids basis (MPC85) as the MF feed. The limiting flux for the MF of diluted MPC85 was determined at 8, 9, and 10% TP concentration in the recirculation loop. The experiment using the 3-mm CD membranes was replicated 3 times for a total of 9 runs. On the morning of each run MPC85 was diluted with reverse osmosis water to a MF feed TP concentration of 5.4%. In all runs the starting flux was 55kg/m(2) per hour, the flux was then increased in steps until the limiting flux was reached. For the 3-mm CD membranes, the limiting flux was 128▒0.3, 109▒4, and 97▒0.5kg/m(2) per hour at recirculation loop TP concentrations of 8.1▒0.07, 9.2▒0.04, and 10.2▒0.03%, respectively. For the 3-mm CD membranes, increasing the flux from the starting to the limiting flux decreased the SP removal factor from 0.72▒0.02 to 0.67▒0.01; however, no difference in SP removal factor among the target recirculation loop TP concentrations was detected. The limiting flux at each recirculation loop target TP concentration was lower for the 3- compared with the 4-mm CD membranes. The differences in limiting fluxes between the 3- and 4-mm CD membranes were explained in part by the difference in cross-flow velocity (5.5▒0.03 and 7.0▒0.03 m/s for the 3- and 4-mm CD membranes, respectively). The SP removal factor was also lower for the 3- compared with the 4-mm CD membranes, indicating that more membrane fouling may have occurred in the 3- versus 4-mm CD membranes. PMID:25892692
Aquaporin3 is a sperm water channel essential for postcopulatory sperm osmoadaptation and migration
Chen, Qi; Peng, Hongying; Lei, Li; Zhang, Ying; Kuang, Haibin; Cao, Yujing; Shi, Qi-xian; Ma, Tonghui; Duan, Enkui
In the journey from the male to female reproductive tract, mammalian sperm experience a natural osmotic decrease (e.g., in mouse, from ?415 mOsm in the cauda epididymis to ?310 mOsm in the uterine cavity). Sperm have evolved to utilize this hypotonic exposure for motility activation, meanwhile efficiently silence the negative impact of hypotonic cell swelling. Previous physiological and pharmacological studies have shown that ion channel-controlled water influx/efflux is actively involved in the process of sperm volume regulation; however, no specific sperm proteins have been found responsible for this rapid osmoadaptation. Here, we report that aquaporin3 (AQP3) is a sperm water channel in mice and humans. Aqp3-deficient sperm show normal motility activation in response to hypotonicity but display increased vulnerability to hypotonic cell swelling, characterized by increased tail bending after entering uterus. The sperm defect is a result of impaired sperm volume regulation and progressive cell swelling in response to physiological hypotonic stress during male-female reproductive tract transition. Time-lapse imaging revealed that the cell volume expansion begins at cytoplasmic droplet, forcing the tail to angulate and form a hairpin-like structure due to mechanical membrane stretch. The tail deformation hampered sperm migration into oviduct, resulting in impaired fertilization and reduced male fertility. These data suggest AQP3 as an essential membrane pathway for sperm regulatory volume decrease (RVD) that balances the ōtrade-offö between sperm motility and cell swelling upon physiological hypotonicity, thereby optimizing postcopulatory sperm behavior. PMID:21135872
Gene structure, cDNA cloning, and expression of a mouse mercurial-insensitive water channel
Ma, T.; Yang, B.; Verkman, A.S. [Univ. of California, San Francisco, CA (United States)] [Univ. of California, San Francisco, CA (United States)
Three cDNAs encoding isoforms of a mercurial-insensitive water channel (mMIWC) were cloned from a mouse brain cDNA library. The predicted proteins had distinct N-terminal sequences and were 32.0 (mMIWC1), 34.3 (mMIWC2), and 37.8 (mMIWC3) kDa. Immunoblot analysis of mouse brain membranes with a C-terminus-derived polyclonal antibody was consistent with the predicted sizes. Expression in Xenopus oocytes indicated that each isoform functioned as a mercurial-insensitive, water-selective channel. Northern blot analysis indicated a major transcript of 5.5 kb in brain > eye > lung {approximately} kidney, and a minor 1.7-kb transcript in heart and muscle. Sequence comparison of mMIWC1 cDNA with a cloned 24-kb mouse genomic DNA indicated three introns (lengths 1.5, 0.5, and 4.0 kb) separating four exons with boundaries at amino acids 127, 182, and 209; analysis of mMIWC2 and mMIWC3 sequences indicated an additional intron at nucleotide -34 upstream from the mMIWC translation initiation site. The mMIWC1 promoter was identified and contained TATA, CAAT, GATA, and AP-2 elements; primer extension revealed mMIWC transcription initiation at 621 bp upstream from the mMIWC1 translational initiation site. Genomic Southern blot analysis revealed a single-copy mMIWC gene. These data indicate the presence of multiple mMIWC isoforms with distinct N-termini encoded by mRNAs produced by distinct transcriptional units and alternative splicing. The genomic cloning of mMIWC represents the first step in the construction of a targeting vector for mMIWC gene knockout. 21 refs., 4 figs.
Ca2+-Calmodulin Modulates Ion Channel Activity in Storage Protein Vacuoles of Barley Aleurone Cells.
Bethke, P. C.; Jones, R. L.
Many plant ion channels have been identified, but little is known about how these transporters are regulated. We have investigated the regulation of a slow vacuolar (SV) ion channel in the tonoplast of barley aleurone storage protein vacuoles (SPV) using the patch-clamp technique. SPV were isolated from barley aleurone protoplasts incubated with CaCl2 in the presence or absence of gibberellic acid (GA) or abscisic acid (ABA). A slowly activating, voltage-dependent ion channel was identified in the SPV membrane. Mean channel conductance was 26 pS when 100 mM KCl was on both sides of the membrane, and reversal potential measurements indicated that most of the current was carried by K+. Treatment of protoplasts with GA3 increased whole-vacuole current density compared to SPV isolated from ABA- or CaCl2-treated cells. The opening of the SV channel was sensitive to cytosolic free Ca2+ concentration ([Ca2+]i) between 600 nM and 100 [mu]M, with higher [Ca2+]i resulting in a greater probability of channel opening. SV channel activity was reduced greater than 90% by the calmodulin (CaM) inhibitors W7 and trifluoperazine, suggesting that Ca2+ activates endogenous CaM tightly associated with the membrane. Exogenous CaM partially reversed the inhibitory effects of W7 on SV channel opening. CaM also sensitized the SV channel to Ca2+. In the presence of ~3.5 [mu]M CaM, specific current increased by approximately threefold at 2.5 [mu]M Ca2+ and by more than 13-fold at 10 [mu]M Ca2+. Since [Ca2+]i and the level of CaM increase in barley aleurone cells following exposure to GA, we suggest that Ca2+ and CaM act as signal transduction elements mediating hormone-induced changes in ion channel activity. PMID:12244238
Isolation and Characterization of an Amino Acid-Selective Channel Protein Present in the Chloroplastic Outer Envelope Membrane
Kai Pohlmeyer; Jurgen Soll; Thomas Steinkamp; Silke Hinnah; Richard Wagner
The reconstituted pea chloroplastic outer envelope protein of 16 kDa (OEP16) forms a slightly cation-selective, high-conductance channel with a conductance of Lambda = 1,2 nS (in 1 M KCl). The open probability of OEP16 channel is highest at 0 mV (Popen = 0.8), decreasing exponentially with higher potentials. Transport studies using reconstituted recombinant OEP16 protein show that the OEP16 channel
Technology Transfer Automated Retrieval System (TEKTRAN)
Aims: To evaluate the effectiveness of continuous separation channel centrifugation for concentrating Toxoplasma gondii and Cyclospora cayetanensis from drinking water and environmental waters.Methods and Results: Ready-to-seed vials with known quantities of Toxoplasma gondii and Cyclospora cayetane...
Design of Peptide-Membrane Interactions to Modulate Single-File Water Transport through Modified Gramicidin Channels
de Groot, Bert
water channels have been proposed as highly potent water filters (14), whereas other channels couldDesign of Peptide-Membrane Interactions to Modulate Single-File Water Transport through Modified Biomolecular Dynamics Group, Max Planck Institute for Biophysical Chemistry, Go©ttingen, Germany ABSTRACT Water
Channel activity of deamidated isoforms of prion protein fragment 106-126 in planar lipid bilayers.
Kourie, J I; Farrelly, P V; Henry, C L
Using the lipid bilayer technique, we have found that age-related derivatives, PrP[106-126] (L-Asp108) and PrP[106-126] (L-iso-Asp108), of the prion protein fragment 106-126 (PrP[106-126] (Asn108)) form heterogeneous ion channels. The deamidated isoforms, PrP[106-126] (L-Asp108) and PrP[106-126] (L-iso-Asp108), showed no enhanced propensity to form heterogeneous channels compared with PrP[106-126] (Asn108). One of the PrP[106-126] (L-Asp108)- and PrP[106-126] (L-iso-Asp108)-formed channels had three kinetic modes. The current-voltage (I-V) relationship of this channel, which had a reversal potential, E(rev), between -40 and -10 mV close to the equilibrium potential for K+ (E(K)-35 mV), exhibited a sigmoidal shape. The value of the maximal slope conductance (g(max)) was 62.5 pS at positive potentials between 0 and 140 mV. The probability (P(o)) and the frequency (F(o)) of the channel being open had inverted and bell-shaped curves, respectively, with a peak at membrane potential (V(m)) between -80 and +80 mV. The mean open and closed times (T(o) and T(c)) had inverted bell-shaped curves. The biophysical properties of PrP[106-126] (L-Asp108)- and PrP[106-126] (L-iso-Asp108)-formed channels and their response to Cu(2+) were similar to those of channels formed with PrP[106-126] (Asn108). Cu(2+) shifted the kinetics of the channel from being in the open state to a "burst state" in which rapid channel activities were separated by long durations of inactivity. The action of Cu(2+) on the open channel activity was both time-dependent and voltage-dependent. The fact that Cu(2+) induced changes in the kinetics of this channel with no changes in the conductance of the channel indicated that Cu(2+) binds at the mouth of the channel. Consistently with the hydrophilic and structural properties of PrP[106-126], the Cu(2+)-induced changes in the kinetic parameters of this channel suggest that the Cu(2+) binding site could be located at M(109) and H(111) of this prion fragment. PMID:11592116
Small Heat Shock Protein ?A-crystallin Regulates Epithelial Sodium Channel Expression*
Kashlan, Ossama B.; Mueller, Gunhild M.; Qamar, Mohammad Z.; Poland, Paul A.; Ahner, Annette; Rubenstein, Ronald C.; Hughey, Rebecca P.; Brodsky, Jeffrey L.; Kleyman, Thomas R.
Integral membrane proteins are synthesized on the cytoplasmic face of the endoplasmic reticulum (ER). After being translocated or inserted into the ER, they fold and undergo post-translational modifications. Within the ER, proteins are also subjected to quality control checkpoints, during which misfolded proteins may be degraded by proteasomes via a process known as ER-associated degradation. Molecular chaperones, including the small heat shock protein ?A-crystallin, have recently been shown to play a role in this process. We have now found that ?A-crystallin is expressed in cultured mouse collecting duct cells, where apical Na+ transport is mediated by epithelial Na+ channels (ENaC). ENaC-mediated Na+ currents in Xenopus oocytes were reduced by co-expression of ?A-crystallin. This reduction in ENaC activity reflected a decrease in the number of channels expressed at the cell surface. Furthermore, we observed that the rate of ENaC delivery to the cell surface of Xenopus oocytes was significantly reduced by co-expression of ?A-crystallin, whereas the rate of channel retrieval remained unchanged. We also observed that ?A-crystallin and ENaC co-immunoprecipitate. These data are consistent with the hypothesis that small heat shock proteins recognize ENaC subunits at ER quality control checkpoints and can target ENaC subunits for ER-associated degradation. PMID:17664274
Studying the stoichiometries of membrane proteins by mass spectrometry: microbial rhodopsins and a potassium ion channel.
Hoffmann, Jan; Aslimovska, Lubica; Bamann, Christian; Glaubitz, Clemens; Bamberg, Ernst; Brutschy, Bernd
In the present work we demonstrate the advantages of LILBID mass spectrometry in the mass analysis of membrane proteins with emphasis on ion-pumps and channels. Due to their hydrophobic nature, membrane proteins have to be solubilized by detergents. However, these molecules tend to complicate the analysis by mass spectrometry. In LILBID, detergent molecules are readily tolerated which allows for the study of solution phase quaternary structures of membrane proteins. This is shown for the proton-pump bacteriorhodospin and the potassium channel KcsA where in both cases the stoichiometries found by LILBID reflect the known structures from 2D or 3D crystals. With proteorhodopsin we demonstrate a preliminary detergent screening showing different structures in different detergents and the implications for the functionality of this protein. We show that Triton-X 100 prevents the formation of the pentamer of proteorhodopsin. Furthermore, the quaternary structures of proteorhodopsin cloned without the signal peptide and of the cation channel channelrhodopsin-2 were studied. The intrinsic properties of channelrhodopsin-2 allow for mass spectrometric analysis in very high salt concentrations up to 100 mM of NaCl. In summary we demonstrate that LILBID is an alternative mass spectrometric method for the analysis of membrane proteins from solution phase. PMID:20355288
Expression of aquaporin-4 water channels in the digestive tract of the guinea pig.
Jiang, Ling; Li, Jian; Liu, Xiaofeng; Burnstock, Geoffrey; Xiang, Zhenghua
Expression of the aquaporin-4 (AQP4) water channel was systematically studied in the digestive tract of the guinea pig using Western blot and immunofluorescence techniques. The results showed that AQP4 was expressed widely in different segments of the guinea pig digestive tract. AQP4-immunoreactivity was confined to parietal cells in the stomach, and absorptive and glandular epithelial cells of small and large intestine. AQP4 protein was also expressed by enteric glial cells of submucosal and myenteric ganglia and primary nerve trunks. AQP4 was expressed by both type I and type II enteric gliocytes, but not by type III or type IV enteric gliocytes, indicating that enteric gliocytes have a heterogeneous distribution in the gut wall. In addition, different patterns of AQP4 expression in the enteric nervous system of human, guinea pig, rat and mouse colon mucosa were identified: in rat and mouse AQP4 was localised to a small subpopulation of neurons; in the guinea pig AQP4 was localised to enteric glial cells; and in the human colon mucosa, AQP4 was also detected mainly in the glial cells. It has been speculated that AQP4 may be involved in water transport in the gastrointestinal tract. Its role in enteric neurons and glia is unknown, but, by analogy with the brain, AQP4 may be involved in the formation and resolution of edema. PMID:24122228
Claudins reign: The claudin/EMP/PMP22/? channel protein family in C. elegans.
Simske, Jeffrey S
The claudin family of integral membrane proteins was identified as the major protein component of the tight junctions in all vertebrates. Since their identification, claudins, and their associated pfam00822 superfamily of proteins have been implicated in a wide variety of cellular processes. Claudin homologs have been identified in invertebrates as well, including Drosophila and C. elegans. Recent studies demonstrate that the C. elegans claudins, clc-1-clc- 5, and similar proteins in the greater PMP22/EMP/claudin/voltage-gated calcium channel ? subunit family, including nsy-4, and vab-9, while highly divergent at a sequence level from each other and from the vertebrate claudins, in many cases play roles similar to those traditionally assigned to their vertebrate homologs. These include regulating cell adhesion and passage of small molecules through the paracellular space, channel activity, protein aggregation, sensitivity to pore-forming toxins, intercellular signaling, cell fate specification and dynamic changes in cell morphology. Study of claudin superfamily proteins in C. elegans should continue to provide clues as to how claudin family protein function has been adapted to perform diverse functions at specialized cell-cell contacts in metazoans. PMID:24665403
Isolation and identification of a sodium channel-inhibiting protein from eggs of black widow spiders.
Li, Jianjun; Yan, Yizhong; Yu, Hai; Peng, Xiaozhen; Zhang, Yiya; Hu, Weijun; Duan, Zhigui; Wang, Xianchun; Liang, Songping
The eggs of black widow spider (L. tredecimguttatus) have been demonstrated to be rich in biologically active components that exhibit great research value and application foreground. In the present study, a protein toxin, named Latroeggtoxin-II, was isolated from the eggs using the combination of gel filtration, ion exchange chromatography and reversed-phase high performance liquid chromatography. Electrospray mass spectrometric analysis indicated that the molecular weight of the protein was 28.69 kDa, and Edman degradation revealed that its N-terminal sequence was ESIQT STYVP NTPNQ KFDYE VGKDY-. After being abdominally injected into mice and P. americana, the protein could make the animals especially P. americana display a series of poisoning symptoms. Electrophysiological experiments demonstrated that the protein could selectively inhibit tetrodotoxin-resistant Na(+) channel currents in rat dorsal root ganglion neurons, without significant effect on the tetrodotoxin-sensitive Na(+) channel currents. Using multiple proteomic strategies, the purified protein was shown to have only a few similarities to the existing proteins in the databases, suggesting that it was a novel protein isolated from the eggs of black widow spiders. PMID:24412150
Experimental study of two-phase water flow in vertical thin rectangular channels
NASA Astrophysics Data System (ADS)
Wright, Christopher T.; O'Brien, James E.; Anderson, Elgin A.
An experimental heat transfer study of two-phase water flow in vertical thin rectangular channels with side vents is conducted. A multiple, heated channel configuration with up- and down-flow conditions is investigated. Parallel heated and unheated flow channels test the effects of cross flow on the onset of nucleate boiling (ONB) and critical heat flux (CHF). The test apparatus provides pressure and substrate temperature data and visual data of the boiling regimes and side-vent flow patterns. The objectives are to determine the two-phase, heat and mass transfer characteristics between adjacent channels as permitted by side-vent cross flow. These data will help develop ONB and CHF correlations for flow geometries typical of plate-type nuclear reactors and heat exchangers. Fundamentally, the data shows how the geometry, flow conditions, and channel configurations affect the heat transfer characteristics of interior channel flows, essential in understanding the ONB and CHF phenomena.
Time-Resolved Images of the Decay of the Gas Channel Induced by Pulsed Positive Streamer Discharge in Water
Xiao Qiong Wen; Gui Shi Liu; Zhen Feng Ding
In an underwater streamer discharge, plasma chan- nels are created and propagate in water. After the discharge, the ionized gas channels gradually cool down and decay. In this paper, a time-resolved study on the decay process of the gas channels induced by pulsed positive streamer discharge in water is performed by using a high-speed camera system. The gas channels created
The trafficking protein SYP121 of Arabidopsis connects programmed stomatal closure and K? channel activity with vegetative growth.
Eisenach, Cornelia; Chen, Zhong-Hua; Grefen, Christopher; Blatt, Michael R
The vesicle-trafficking protein SYP121 (SYR1/PEN1) was originally identified in association with ion channel control at the plasma membrane of stomatal guard cells, although stomata of the Arabidopsis syp121 loss-of-function mutant close normally in ABA and high Ca▓?. We have now uncovered a set of stomatal phenotypes in the syp121 mutant that reduce CO? assimilation, slow vegetative growth and increase water use efficiency in the whole plant, conditional upon high light intensities and low relative humidity. Stomatal opening and the rise in stomatal transpiration of the mutant was delayed in the light and following Ca▓?-evoked closure, consistent with a constitutive form of so-called programmed stomatal closure. Delayed reopening was observed in the syp121, but not in the syp122 mutant lacking the homologous gene product; the delay was rescued by complementation with wild-type SYP121 and was phenocopied in wild-type plants in the presence of the vesicle-trafficking inhibitor Brefeldin A. K? channel current that normally mediates K? uptake for stomatal opening was suppressed in the syp121 mutant and, following closure, its recovery was slowed compared to guard cells of wild-type plants. Evoked stomatal closure was accompanied by internalisation of GFP-tagged KAT1 K? channels in both wild-type and syp121 mutant guard cells, but their subsequently recycling was slowed in the mutant. Our findings indicate that SYP121 facilitates stomatal reopening and they suggest that K? channel traffic and recycling to the plasma membrane underpins the stress memory phenomenon of programmed closure in stomata. Additionally, they underline the significance of vesicle traffic for whole-plant water use and biomass production, tying SYP121 function to guard cell membrane transport and stomatal control. PMID:21914010
Gating of the TrkH Ion Channel by its Associated RCK Protein, Trka
Cao, Yu; Pan, Yaping; Huang, Hua; Jin, Xiangshu; Levin, Elena J.; Kloss, Brian; Zhou, Ming
TrkH belongs to a superfamily of K+ transport proteins required for growth of bacteria in low external K+ concentrations. The crystal structure of TrkH from Vibrio parahaemolyticus showed that TrkH resembles a K+ channel, and may have a gating mechanism substantially different from K+ channels. TrkH assembles with TrkA, a cytosolic protein comprising two Regulate-the-Conductance-of-K+, or RCK domains, which are found in certain K+ channels and control their gating. However, fundamental questions on whether TrkH is an ion channel and how it is regulated by TrkA remain unresolved. Here we show single-channel activity of TrkH that is upregulated by ATP via TrkA. We report two structures of the tetrameric TrkA ring, one in complex with TrkH and one in isolation, in which the ring assumes two dramatically different conformations. These results suggest a mechanism for how ATP increases TrkH activity by inducing conformational changes in TrkA. PMID:23598339
Viral M2 ion channel protein: a promising target for anti-influenza drug discovery.
Moorthy, N S Hari Narayana; Poongavanam, Vasanthanathan; Pratheepa, V
Influenza virus is an important RNA virus causing pandemics (Spanish Flu (1918), Asian Flu (1957), Hong Kong Flu (1968) and Swine Flu (2009)) over the last decades. Due to the spontaneous mutations of these viral proteins, currently available antiviral and anti-influenza drugs quickly develop resistance. To account this, only limited antiinfluenza drugs have been approved for the therapeutic use. These include amantadine and rimantadine (M2 proton channel blockers), zanamivir, oseltamivir and peramivir (neuraminidase inhibitors), favipravir (polymerase inhibitor) and laninamivir. This review provides an outline on the strategies to develop novel, potent chemotherapeutic agents against M2 proton channel. Primarily, the M2 proton channel blockers elicit pharmacological activity through destabilizing the helices by blocking the proton transport across the transmembrane. The biologically important compounds discovered using the scaffolds such as bisnoradmantane, noradamantane, triazine, spiroadamantane, isoxazole, amino alcohol, azaspiro, spirene, pinanamine, etc are reported to exhibit anti-influenza activity against wild or mutant type (S31N and V27A) of M2 proton channel protein. The reported studies explained that the adamantane based compounds (amantadine and rimantadine) strongly interact with His37 (through hydrogen bonding) and Ala30, Ile33 and Gly34 residues (hydrophobic interactions). The adamantane and the non-adamantane scaffolds fit perfectly in the active site pocket present in the wild type and the charged amino groups (ammonium) create positive electrostatic potential, which blocks the transport of protons across the pore. In the mutated proteins, larger or smaller binding pocket are created by small or large mutant residues, which do not allow the molecules fit in the active site. This causes the channel to be unblocked and the protons are allowed to transfer inside the pore. The structural analysis of the M2 proton channel blockers illustrated that the adamantane derivatives have action against both influenza A and B, but have no effect on the mutants. PMID:25342196
33 CFR 207.640 - Sacramento Deep Water Ship Channel Barge Lock and Approach Canals; use, administration, and...
Code of Federal Regulations, 2011 CFR
33 Navigation and Navigable Waters 3 2011-07-01 2011-07-01 false Sacramento Deep Water Ship Channel Barge Lock and Approach Canals...Section 207.640 Navigation and Navigable Waters CORPS OF ENGINEERS, DEPARTMENT OF...
33 CFR 207.640 - Sacramento Deep Water Ship Channel Barge Lock and Approach Canals; use, administration, and...
Code of Federal Regulations, 2013 CFR
33 Navigation and Navigable Waters 3 2013-07-01 2013-07-01 false Sacramento Deep Water Ship Channel Barge Lock and Approach Canals...Section 207.640 Navigation and Navigable Waters CORPS OF ENGINEERS, DEPARTMENT OF...
Palmitoylation and Membrane Association of the Stress Axis Regulated Insert (STREX) Controls BK Channel Regulation by Protein Kinase C*
Zhou, Xiaobo; Wulfsen, Iris; Korth, Michael; McClafferty, Heather; Lukowski, Robert; Shipston, Michael J.; Ruth, Peter; Dobrev, Dobromir; Wieland, Thomas
Large-conductance, calcium- and voltage-gated potassium (BK) channels play an important role in cellular excitability by controlling membrane potential and calcium influx. The stress axis regulated exon (STREX) at splice site 2 inverts BK channel regulation by protein kinase A (PKA) from stimulatory to inhibitory. Here we show that palmitoylation of STREX controls BK channel regulation also by protein kinase C (PKC). In contrast to the 50% decrease of maximal channel activity by PKC in the insertless (ZERO) splice variant, STREX channels were completely resistant to PKC. STREX channel mutants in which Ser700, located between the two regulatory domains of K+ conductance (RCK) immediately downstream of the STREX insert, was replaced by the phosphomimetic amino acid glutamate (S700E) showed a ?50% decrease in maximal channel activity, whereas the S700A mutant retained its normal activity. BK channel inhibition by PKC, however, was effectively established when the palmitoylation-mediated membrane-anchor of the STREX insert was removed by either pharmacological inhibition of palmitoyl transferases or site-directed mutagenesis. These findings suggest that STREX confers a conformation on BK channels where PKC fails to phosphorylate and to inhibit channel activity. Importantly, PKA which inhibits channel activity by disassembling the STREX insert from the plasma membrane, allows PKC to further suppress the channel gating independent from voltage and calcium. Our results present an important example for the cross-talk between ion channel palmitoylation and phosphorylation in regulation of cellular excitability. PMID:22843729
Determining the water sorption monolayer of lyophilized pharmaceutical proteins.
Costantino, H R; Curley, J G; Hsu, C C
The concept of monolayer water coverage is useful in the development of lyophilized protein formulations. Herein, we have explored three different methodologies to determine the water monolayer for pharmaceutical proteins: (1) theoretical prediction based on the amino acid composition and their relative propensities to sorb water; (2) a traditional adsorption isotherm measurement by Karl Fischer water titration of samples held at various relative humidities (created by saturated salt solutions); and (3) an adsorption isotherm measurement with a gravimetric sorption analyzer (GSA), which consists of a microbalance within a computer-controlled humidified environment. Data from the latter two methods were analyzed with the Brunauer-Emmett-Teller (BET) gas adsorption equation to yield experimental monolayers. In our study, we examined six different therapeutic proteins and found that for each case all three approaches yielded similar results for the water monolayer. We also attempted to use the BET equation to determine the water monolayer for a model sugar (trehalose) and polyol (mannitol), which are potential excipients in pharmaceutical protein formulations. We found that calculations from the data obtained by the traditional and GSA methods yielded consistent results for trehalose, which remained amorphous upon lyophilization. Mannitol tended to form anhydrous crystals upon freezedrying, and was thus not amenable to analysis. The utility of both traditional and GSA methods for determining the water monolayer was extended to colyophilized protein:sugar systems as well. PMID:9423152
Mapping hydration dynamics and coupled water-protein fluctuations around a protein surface
NASA Astrophysics Data System (ADS)
Zhang, Luyuan; Wang, Lijuan; Kao, Ya-Ting; Qiu, Weihong; Yang, Yi; Okobiah, Oghaghare; Zhong, Dongping
Elucidation of the molecular mechanism of water-protein interactions is critical to understanding many fundamental aspects of protein science, such as protein folding and misfolding and enzyme catalysis. We recently carried out a global mapping of protein-surface hydration dynamics around a globular ?-helical protein apomyoglobin. The intrinsic optical probe tryptophan was employed to scan the protein surface one at a time by site-specific mutagenesis. With femtosecond resolution, we mapped out the dynamics of water-protein interactions with more than 20 mutants and for two states, native and molten globular. A robust bimodal distribution of time scales was observed, representing two types of water motions: local relaxation and protein-coupled fluctuations. The time scales show a strong correlation with the local protein structural rigidity and chemical identity. We also resolved two distinct contributions to the overall Stokes-shifts from the two time scales. These results are significant to understanding the role of hydration water on protein structural stability, dynamics and function.
Effect of channel catfish stocking rate on yield and water quality in an intensive production system
Technology Transfer Automated Retrieval System (TEKTRAN)
The effect of stocking rate on production of NWAC 103 strain channel catfish (Ictalurus punctatus) and water quality was investigated using a completely randomized design in an intensively managed biofloc raceway system. Each of the 9 HDPE-lined raceways (4.6 m x 9.2 m with a 0.9-m water depth; 42.2...
Flow cytometry and sorting of amphibian bladder endocytic vesicles containing ADH-sensitive water channels
F. G. Goot; A. Seigneur; J.-C. Gaucher; P. Ripoche
The water permeability of ADH target epithelial cells is believed to be regulated by a cycle of exo-endocytosis of vesicles containing functional water channels. These vesicles were selectively labeled in intact frog urinary bladders with an impermeant fluorescent marker, 6-carboxyfluorescein. Vesicle suspensions containing the labeled endosomes were obtained by homogenization and differential centrifugation of bladder epithelial cells. The osmotic permeability
Formation of Mangala Valles outflow channel, Mars: Morphological development and water discharge and duration estimates
Harald J. Leask; Lionel Wilson; Karl L. Mitchell
The morphology of features on the floor of the Mangala Valles suggests that the channel system was not bank-full for most of the duration of its formation by water being released from its source, the Mangala Fossa graben. For an estimated typical 50 m water depth, local slopes of sin ? = ?0.002 imply a discharge of ?1 ū 107
CROSS-DISCIPLINARY PHYSICS AND RELATED AREAS OF SCIENCE AND TECHNOLOGY: Enhancement of water permeation across nanochannels by partial charges mimicked from biological channels
NASA Astrophysics Data System (ADS)
Gong, Xiao-Jing; Fang, Hai-Ping
In biological water channel aquaporins (AQPs), it is believed that the bipolar orientation of the single-file water molecules inside the channel blocks proton permeation but not water transport. In this paper, the water permeation and particularly the water-selective behaviour across a single-walled carbon nanotube (SWNT) with two partial charges adjacent to the wall of the SWNT are studied by molecular dynamics simulations, in which the distance between the two partial charges is varied from 0.14 nm to 0.5 nm and the charges each have a quantity of 0.5 e. The two partial charges are used to mimic the charge distribution of the conserved non-pseudoautosomal (NPA) (asparagine/proline/alanine) regions in AQPs. Compared with across the nanochannel in a system with one +1 e charge, the water permeation across the nanochannel is greatly enhanced in a system with two +0.5 e charges when charges are close to the nanotube, i.e. the two partial charges permit more rapid water diffusion and maintain better bipolar order along the water file when the distance between the two charges and the wall of SWNT is smaller than about 0.05 nm. The bipolar orientation of the single-file water molecules is crucial for the exclusion of proton transfer. These findings may serve as guidelines for the future nanodevices by using charges to transport water and have biological implications because membrane water channels share a similar single-file water chain and positive charged region at centre and provide an insight into why two residues are necessitated in the central region of water channel protein.
Impacts of commercial navigation on water quality in the Illinois River Channel. Research report
Butts, T.A.; Shackleford, D.B.
The U.S. Army Corps of Engineers shut down the navigation locks at the LaGrange and Peoria dams on the Illinois River for repairs for 58 days during summer 1987, which prevented commercial tows from traversing a 151-mile reach of the waterway. A study was designed to collect water quality and benthic data from the main channel during the shutdown and for 30-day periods before and after it. Statistical tests were used to determine whether water quality conditions improved when commercial navigation was absent. The results indicated that, at the present rate, commercial traffic does not significantly change overall water and benthic sediment quality in the channel.
Lipid bilayer regulation of membrane protein function: gramicidin channels as molecular force probes
Lundbµk, Jens A.; Collingwood, Shemille A.; Ing¾lfsson, Helgi I.; Kapoor, Ruchi; Andersen, Olaf S.
Membrane protein function is regulated by the host lipid bilayer composition. This regulation may depend on specific chemical interactions between proteins and individual molecules in the bilayer, as well as on non-specific interactions between proteins and the bilayer behaving as a physical entity with collective physical properties (e.g. thickness, intrinsic monolayer curvature or elastic moduli). Studies in physico-chemical model systems have demonstrated that changes in bilayer physical properties can regulate membrane protein function by altering the energetic cost of the bilayer deformation associated with a protein conformational change. This type of regulation is well characterized, and its mechanistic elucidation is an interdisciplinary field bordering on physics, chemistry and biology. Changes in lipid composition that alter bilayer physical properties (including cholesterol, polyunsaturated fatty acids, other lipid metabolites and amphiphiles) regulate a wide range of membrane proteins in a seemingly non-specific manner. The commonality of the changes in protein function suggests an underlying physical mechanism, and recent studies show that at least some of the changes are caused by altered bilayer physical properties. This advance is because of the introduction of new tools for studying lipid bilayer regulation of protein function. The present review provides an introduction to the regulation of membrane protein function by the bilayer physical properties. We further describe the use of gramicidin channels as molecular force probes for studying this mechanism, with a unique ability to discriminate between consequences of changes in monolayer curvature and bilayer elastic moduli. PMID:19940001
Hydration water dynamics and instigation of protein structuralrelaxation
Russo, Daniela; Hura, Greg; Head-Gordon, Teresa
Until a critical hydration level is reached, proteins do not function. This critical level of hydration is analogous to a similar lack of protein function observed for temperatures below a dynamical temperature range of 180-220K that also is connected to the dynamics of protein surface water. Restoration of some enzymatic activity is observed in partially hydrated protein powders, sometimes corresponding to less than a single hydration layer on the protein surface, which indicates that the dynamical and structural properties of the surface water is intimately connected to protein stability and function. Many elegant studies using both experiment and simulation have contributed important information about protein hydration structure and timescales. The molecular mechanism of the solvent motion that is required to instigate the protein structural relaxation above a critical hydration level or transition temperature has yet to be determined. In this work we use experimental quasi-elastic neutron scattering (QENS) and molecular dynamics simulation to investigate hydration water dynamics near a greatly simplified protein system. We consider the hydration water dynamics near the completely deuterated N-acetyl-leucine-methylamide (NALMA) solute, a hydrophobic amino acid side chain attached to a polar blocked polypeptide backbone, as a function of concentration between 0.5M-2.0M under ambient conditions. We note that roughly 50-60% of a folded protein's surface is equally distributed between hydrophobic and hydrophilic domains, domains whose lengths are on the order of a few water diameters, that justify our study of hydration dynamics of this simple model protein system. The QENS experiment was performed at the NIST Center for Neutron Research, using the disk chopper time of flight spectrometer (DCS). In order to separate the translational and rotational components in the spectra, two sets of experiments were carried out using different incident neutron wavelengths of 7.5{angstrom} and 5.5{angstrom} to give two different time resolutions. All the spectra have been measure at room temperature. The spectra were corrected for the sample holder contribution and normalized using the vanadium standard. The resulting data were analyzed with DAVE programs (http://www.ncnr.nist.gov/dave/). The AMBER force field and SPCE water model were used for modeling the NALMA solute and water, respectively. For the analysis of the water dynamics in the NALMA aqueous solutions, we performed simulations of a dispersed solute configuration consistent with our previous structural analysis, where we had primarily focused on the structural organization of these peptide solutions and their connection to protein folding. Further details of the QENS experiment and molecular dynamics simulations are reported elsewhere.
More than one dynamic crossover in protein hydration water
Marco G. Mazza; Kevin Stokely; Sara E. Pagnotta; Fabio Bruni; H. Eugene Stanley; Giancarlo Franzese
Studies of liquid water in its supercooled region have led to many insights into the structure and behavior of water. While bulk water freezes at its homogeneous nucleation temperature of approximately 235 K, for protein hydration water, the binding of water molecules to the protein avoids crystallization. Here we study the dynamics of the hydrogen bond (HB) network of a percolating layer of water molecules, comparing measurements of a hydrated globular protein with the results of a coarse-grained model that has been shown to successfully reproduce the properties of hydration water. With dielectric spectroscopy we measure the temperature dependence of the relaxation time of protons charge fluctuations. These fluctuations are associated to the dynamics of the HB network of water molecules adsorbed on the protein surface. With Monte Carlo (MC) simulations and mean--field (MF) calculations we study the dynamics and thermodynamics of the model. In both experimental and model analyses we find two dynamic crossovers: (i) one at about 252 K, and (ii) one at about 181 K. The agreement of the experiments with the model allows us to relate the two crossovers to the presence of two specific heat maxima at ambient pressure. The first is due to fluctuations in the HB formation, and the second, at lower temperature, is due to the cooperative reordering of the HB network.
The threshold of vapor channel formation in water induced by pulsed CO2 laser
NASA Astrophysics Data System (ADS)
Guo, Wenqing; Zhang, Xianzeng; Zhan, Zhenlin; Xie, Shusen
Water plays an important role in laser ablation. There are two main interpretations of laser-water interaction: hydrokinetic effect and vapor phenomenon. The two explanations are reasonable in some way, but they can't explain the mechanism of laser-water interaction completely. In this study, the dynamic process of vapor channel formation induced by pulsed CO2 laser in static water layer was monitored by high-speed camera. The wavelength of pulsed CO2 laser is 10.64 um, and pulse repetition rate is 60 Hz. The laser power ranged from 1 to 7 W with a step of 0.5 W. The frame rate of high-speed camera used in the experiment was 80025 fps. Based on high-speed camera pictures, the dynamic process of vapor channel formation was examined, and the threshold of vapor channel formation, pulsation period, the volume, the maximum depth and corresponding width of vapor channel were determined. The results showed that the threshold of vapor channel formation was about 2.5 W. Moreover, pulsation period, the maximum depth and corresponding width of vapor channel increased with the increasing of the laser power.
Partitioning of Water Discharge by Distributary Channels in the Prograding, Wax Lake Delta, Coastal Louisiana, USA
NASA Astrophysics Data System (ADS)
Buttles, J.; Mohrig, D.; Nittrouer, J.; McElroy, B.; Baitis, E.; Allison, M.; Paola, C.; Parker, G.; Kim, W.
How water and sediment is routed through distributary networks on river deltas is incompletely known and a topic of much active research. We have undertaken a study to determine the controls on partitioning of water and sediment discharge in distributary channels of the Wax Lake Delta and to connect these transport processes to the land building associated with the growth of islands that separate distributary channels from each other. Here we present first results from the field project that defines how water from the upstream primary channel is partitioned between the first set of five distributary channels. Measurements of water discharge and channel bathymetry were collected using a 22-ft research vessel equipped with an acoustic Doppler velocity profiler, a swath bathymetry profiler and dual differential GPS antennas. Wax Lake Delta is situated at the downstream end of Wax Lake Outlet, a man-made channel that diverts water and sediment from the lower Atchafalaya River, roughly 20 km upstream from Morgan City, LA. The subaerial delta has been building out into Atchafalaya Bay since roughly 1973 with a delta-front advance rate of about 0.27 km/yr. Associated with this growth has been development of a distributary network of channels that continues to evolve as the delta progrades seaward. Measurements collected in May, 2007 define properties of the upstream channel and the first set of five distributary channels. Characteristic width, depth and water discharge for the upstream channel are 420 m, 21.2 m, and 2900 m3/s. Characteristic values for width, depth and water discharge for the five distributary channels are 1) 270 m, 6.7 m, and 310 m3/s, 2) 300 m, 6.5 m, and 350 m3/s, 3) 650 m, 6.8 m, and 820 m3/s, 4) 395 m, 6.5 m, and 560 m3/s, and 5) 440 m, 6.0 m, and 440 m3/s. These data highlight a number of interesting points regarding the initial set of bifurcations. First, the transition from one to five channels is associated with a two-thirds reduction in characteristic flow depth and one-third reduction in characteristic flow velocity. Measured discharge in the five channels sums to 86% of the upstream channel. Based on field observations we propose that the remaining 14% is associated with shallow flow onto and across the weakly emergent islands. There is very little difference in characteristic channel depth even though widths of the five channels vary by greater than a factor of two. There is also very little difference in the spatially average velocity for the five channels; velocities range between 0.17-0.22 m/s. All of these channel properties will be discussed in the context of sediment transport and sedimentation within this active delta.
Modeling Meandering Channel by Two-Dimensional Shallow Water Equations
NASA Astrophysics Data System (ADS)
Yu, C.; Duan, J. G.
This research is to simulate the process of channel meandering using a two-dimensional depth-averaged hydrodynamic model. The multiple interactions between unsteady flow, turbulence, secondary flow, nonequilibrium sediment transport and bank erosion are considered by the model. The governing equations are the 2D depth-averaged Reynolds-averaged Navier-Stokes (2D-RANS) equations and the Exner equation for bed elevation evolution. The Reynolds stresses are calculated by the k-? turbulence model. The secondary flow, is modeled by the dispersion terms in momentum equations. The spatial lag between the instantaneous flow properties and the rate of sediment transport is simulated by the nonequilibrium sediment transport model. During the process of adaptation, the sediment transport rate gradually develops into the transport capacity of a given flow condition. The evolution of channel bed and bank is modeled by the general Exner equation that accounts for both vertical deformation of bed elevation as well as lateral migration of bank. The system of governing equations is solved by a semi-implicit finite volume method over the Cartesian mesh. The advective fluxes across each cell interface are simultaneously calculated by the extended HLL Riemann solver. At each time step, the diffusion terms in the governing equations are solved by the implicit Euler scheme. The source terms are discretized in a well-balanced way to retain the C-property of the proposed model. Application of the model to different test cases indicates that the model can correctly simulate different phases of meandering channel evolution which include streamwise migration, transverse migration and rotation of channel bends.
Disruption of the Arabidopsis thaliana inward-rectifier K+ channel AKT1 improves plant responses to water stress.
Nieves-Cordones, Manuel; Caballero, Fernando; MartĒnez, Vicente; Rubio, Francisco
The Arabidopsis thaliana inward-rectifier K(+) channel AKT1 plays an important role in root K(+) uptake. Recent results show that the calcineurin B-like (CBL)-interacting protein kinase (CIPK) 23-CBL1/9 complex activates AKT1 in the root to enhance K(+) uptake. In addition, this CIPK-CBL complex has been demonstrated to regulate stomatal movements and plant transpiration. However, a role for AKT1 in plant transpiration has not yet been demonstrated. Here we show that disruption of AKT1 conferred an enhanced response to water stress in plants. Experiments performed in hydroponics showed that, when water potential was diminished by adding polyethylene glycol, akt1 adult plants lost less water than wild-type (WT) plants. Under long-term water stress in soil, adult akt1 plants displayed lower transpiration and less water consumption than WT plants. Finally, akt1 stomata closed more efficiently in response to ABA. Such results were also observed in cipk23 plants. The similar responses shown by cipk23 and akt1 plants to water stress denote that the regulation of AKT1 by CIPK23 may also take place in stomata and has a negative impact on plant performance under water stress conditions. PMID:22210899
Water and Backbone Dynamics in a Hydrated Protein
Diakova, Galina; Goddard, Yanina A.; Korb, Jean-Pierre; Bryant, Robert G.
Abstract Rotational immobilization of proteins permits characterization of the internal peptide and water molecule dynamics by magnetic relaxation dispersion spectroscopy. Using different experimental approaches, we have extended measurements ofĀthe magnetic field dependence of the proton-spin-lattice-relaxation rate by one decade from 0.01 to 300 MHz for 1H and showed that the underlying dynamics driving the protein 1H spin-lattice relaxation is preserved over 4.5 decades in frequency. This extension is critical to understanding the role of 1H2O in the total proton-spin-relaxation process. The fact that the protein-proton-relaxation-dispersion profile is a power law in frequency with constant coefficient and exponent over nearly 5Ādecades indicates that the characteristics of the native protein structural fluctuations that cause proton nuclear spin-lattice relaxation are remarkably constant over this wide frequency and length-scale interval. Comparison of protein-proton-spin-lattice-relaxation rate constants in protein gels equilibrated with 2H2O rather than 1H2O shows that water protons make an important contribution to the total spin-lattice relaxation in the middle of this frequency range for hydrated proteins because of water molecule dynamics in the time range of tens of ns. This water contribution is with the motion of relatively rare, long-lived, and perhaps buried water molecules constrained by the confinement. The presence of water molecule reorientational dynamics in the tens of ns range that are sufficient to affect the spin-lattice relaxation driven by 1H dipole-dipole fluctuations should make the local dielectric properties in the protein frequency dependent in a regime relevant to catalytically important kinetic barriers to conformational rearrangements. PMID:20085726
Preparation of semi-solid aluminum alloy slurry poured through a water-cooled serpentine channel
NASA Astrophysics Data System (ADS)
Chen, Zheng-Zhou; Mao, Wei-Min; Wu, Zong-Chuang
A water-cooled serpentine channel pouring process was invented to produce semi-solid A356 aluminum alloy slurry for rheocasting, and the effects of pouring temperature and circulating cooling water flux on the microstructure of the slurry were investigated. The results show that at the pouring temperature of 640-680░C and the circulating cooling water flux of 0.9 m3/h, the semi-solid A356 aluminum alloy slurry with spherical primary ?(Al) grains can be obtained, whose shape factors are between 0.78 and 0.86 and the grain diameter can reach 48-68 ?m. When the pouring temperatures are at 660-680░C, only a very thin solidified shell remains inside the serpentine channel and can be removed easily. When the serpentine channel is cooled with circulating water, the microstructure of the semi-solid slurry can be improved, and the serpentine channel is quickly cooled to room temperature after the completion of one pouring. In terms of the productivity of the special equipment, the water-cooled serpentine channel is economical and efficient.
Role of water in Protein Aggregation and Amyloid Polymorphism
D. Thirumalai; Govardhan Reddy; John E. Straub
A variety of neurodegenerative diseases are associated with the formation of amyloid plaques. Our incomplete understanding of this process underscores the need to decipher the principles governing protein aggregation. Most experimental and simulation studies have been interpreted largely from the perspective of proteins: the role of solvent has been relatively overlooked. In this Account, we provide a perspective on how interactions with water affect folding landscapes of A$\\beta$ monomers, A$\\beta_{16-22}$ oligomer formation, and protofilament formation in a Sup35 peptide. Simulations show that the formation of aggregation-prone structures (N$^*$) similar to the structure in the fibril requires overcoming high desolvation barrier. The mechanism of protofilament formation in a polar Sup35 peptide fragment illustrates that water dramatically slows down self-assembly. Release of water trapped in the pores as water wires creates protofilament with a dry interface. Similarly, one of the main driving force for addition of a solvated monomer to a preformed fibril is the entropy gain of released water. We conclude by postulating that two-step model for protein crystallization must also hold for higher order amyloid structure formation starting from N$^*$. Multiple N$^*$ structures with varying water content results in a number of distinct water-laden polymorphic structures. In predominantly hydrophobic sequences, water accelerates fibril formation. In contrast, water-stabilized metastable intermediates dramatically slow down fibril growth rates in hydrophilic sequences.
An SF6 Tracer Study of the Flow Dynamics in the Stockton Deep Water Ship Channel: Implications
Ho, David
An SF6 Tracer Study of the Flow Dynamics in the Stockton Deep Water Ship Channel: Implications6) tracer release experi- ment was conducted in the Stockton Deep Water Ship Channel (DWSC concentrations maintained a steady state value of 4 mg l-1 . These values are below water quality objectives
Modulation of reconstituted ATP-sensitive K+ channels by GTP-binding proteins in a mammalian cell line
Sßnchez, Jorge A; Gonoi, Tohru; Inagaki, Nobuya; Katada, Toshiaki; Seino, Susumu
The action of GTP-binding proteins on ATP-sensitive potassium (KATP) channels was investigated. KATP channels were expressed in a mammalian cell line (COS-1 cells) by cotransfecting vectors carrying the sulphonylurea receptor (SUR1) and BIR (Kir6.2), a member of the inward rectifier K+ channel family. G proteins were also tested on KATP channels composed of an isoform of SUR1, SUR2A, in combination with Kir6.2.The ? and ?? subunits of the GTP binding protein Gi were tested separately in inside-out patches under continuous recording. G?-i1 increases the activity of SUR1-Kir6.2 and SUR2A-Kir6.2 channels by 200 and by 30 %, respectively.G?-i2 does not increase the activity of SUR1-Kir6.2 channels, but increases the activity of SUR2A-Kir6.2 channels by 30 %.Control experiments showed that GTP?S, a specific activator of G proteins, and heat- inactivated G?-i1 do not increase the single channel activity.No effects of the other subunits (??) from either Gi1 or Gi2 on the single channel activity were observed.The protein kinase C inhibitors H7 and an inhibitory peptide (FARKGALRQKNV) had no effect on the modulatory action of G?-i1 on SUR1-Kir6.2 channels.We conclude that both types of reconstituted KATP channels are modulated by G proteins. PMID:9518695
Bioinformatic Characterization of the Trimeric Intracellular Cation-Specific Channel Protein Family
Silverio, Abe L. F.
Trimeric intracellular cation-specific (TRIC) channels are integral to muscle excitation¢contraction coupling. TRIC channels provide counter-ionic flux when calcium is rapidly transported from intracellular stores to the cell cytoplasm. Until recently, knowledge of the presence of these proteins was limited to animals. We analyzed the TRIC family and identified a profusion of prokaryotic family members with topologies and motifs similar to those of their eukaryotic counterparts. Prokaryotic members far outnumber eukaryotic members, and although none has been functionally characterized, the evidence suggests that they function as secondary carriers. The presence of fused N- or C-terminal domains of known biochemical functions as well as genomic context analyses provide clues about the functions of these prokaryotic homologs. They are proposed to function in metabolite (e.g., amino acid/ nucleotide) efflux. Phylogenetic analysis revealed that TRIC channel homologs diverged relatively early during evolutionary history and that horizontal gene transfer was frequent in prokaryotes but not in eukaryotes. Topological analyses of TRIC channels revealed that these proteins possess seven putative transmembrane segments (TMSs), which arose by intragenic duplication of a three-TMS polypeptide-encoding genetic element followed by addition of a seventh TMS at the C terminus to give the precursor of all current TRIC family homologs. We propose that this family arose in prokaryotes. PMID:21519847
Mutation of a putative S-nitrosylation site of TRPV4 protein facilitates the channel activates
Lee, Eun Jeoung; Shin, Sung Hwa; Hyun, Sunghee; Chun, Jaesun; Kang, Sang Sun
The transient receptor potential vanilloid 4 (TRPV4) cation channel, a member of the TRP vanilloid subfamily, is expressed in a broad range of tissues. Nitric oxide (NO) as a gaseous signal mediator shows a variety of important biological effects. In many instances, NO has been shown to exhibit its activities via a protein S-nitrosylation mechanism in order to regulate its protein functions. With functional assays via site-directed mutagenesis, we demonstrate herein that NO induces the S-nitrosylation of TRPV4 Ca2+ channel on the Cys853 residue, and the S-nitrosylation of Cys853 reduced its channel sensitivity to 4-? phorbol 12,13-didecanoate and the interaction between TRPV4 and calmodulin. A patch clamp experiment and Ca2+ image analysis show that the S-nitrosylation of Cys853 modulates the TRPV4 channel as an inhibitor. Thus, our data suggest a novel regulatory mechanism of TRPV4 via NO-mediated S-nitrosylation on its Cys853 residue. PMID:21837266
Water flux in membrane fuel cell humidifiers: Flow rate and channel location effects
P. Cave; W. Mķrida
A straight, single channel membrane humidifier was constructed to measure temperature and moisture profiles along both the donor and receiver channels. A persulfonic Nafion membrane was used as the water exchange medium.We report on results obtained with single-phase vapour-to-vapour, counter flow operation. First, the heat loss to the surroundings was quantified and found to affect the overall performance significantly. Second,
Boiling Water Reactor Fuel Cycle Optimization for Prevention of Channel-Blade Interference
Kropaczek, David J.; Karve, Atul A.; Oyarzun, Christian C.; Asgari, Mehdi [Global Nuclear Fuel - Americas, P.O. Box 780, M/C F12, Wilmington, N.C. 28402 (United States); Tusar, James J. [Exelon Corporation, 200 Exelon Way, KSA 2-N, Kennett Square, PA 19348 (United States)
A formal optimization method for eliminating the potential of Boiling Water Reactor channel-blade interference is presented within the context of fuel cycle design. The method is based on the use of threshold constraints on blade force as penalty terms within an objective function that are employed as part of a search algorithm. Results demonstrate the effectiveness of the constraint formulation in eliminating channel-blade interference as part of the design of the core loading and operational strategy. (authors)
Controlled protein adsorption on microfluidic channels with engineered roughness and wettability
Katerina Tsougeni; Panagiota S. Petrou; Dimitris P. Papageorgiou; Sotirios E. Kakabakos; Angeliki Tserepi; Evangelos Gogolides
Plasma processing is demonstrated as a generic technology not only to fabricate, roughen, and control the wetting properties of microfluidic devices but also to control the protein adsorption in microfluidic channels intended for bio-analysis. After lithography on poly(methyl methacrylate) (PMMA) substrates, deep anisotropic O2 plasma etching was utilized to pattern microchannels, at conditions where very rough bottom walls were obtained.
TMEM16A Protein: A New Identity for Ca2+-Dependent Cl? Channels
NSDL National Science Digital Library
Loretta Ferrera (Istituto Giannina Gaslini)
Ca+-dependent Cl? channels (CaCCs) play a variety of physiological roles in different organs and tissues, including transepithelial Cl? secretion, smooth muscle contraction, regulation of neuronal excitability, and transduction of sensory stimuli. The recent identification of TMEM16A protein as an important component of CaCCs should allow a better understanding of their physiological role, structure-function relationship, and regulatory mechanisms.
Investigating reaction pathways in rare events simulations of antibiotics diffusion through protein channels
Eric Hajjar; Amit Kumar; Paolo Ruggerone; Matteo Ceccarelli
In Gram-negative bacteria, outer-membrane protein channels, such as OmpF of Escherichia coli, constitute the entry point of\\u000a various classes of antibiotics. While antibacterial research and development is declining, bacterial resistance to antibiotics\\u000a is rising and there is an emergency call for a new way to develop potent antibacterial agents and to bring them to the market\\u000a faster and at reduced
Water and Proton Conduction through Carbon Nanotubes as Models for Biological Channels
Zhu, Fangqiang; Schulten, Klaus
Carbon nanotubes, unmodified (pristine) and modified through charged atoms, were simulated in water, and their water conduction rates determined. The conducted water inside the nanotubes was found to exhibit a strong ordering of its dipole moments. In pristine nanotubes the water dipoles adopt a single orientation along the tube axis with a low flipping rate between the two possible alignments. Modification can induce in nanotubes a bipolar ordering as previously observed in biological water channels. Network thermodynamics was applied to investigate proton conduction through the nanotubes. PMID:12829479
Dynamics of channel incision in a granular bed driven by subsurface water flow
A. E. Lobkovsky; B. Smith; A. Kudrolli; D. H. Rothman
We propose a dynamical model for the erosive growth of a channel in a granular medium driven by subsurface water flow. The model is inferred from experimental data acquired with a laser-aided imaging technique. The evolution equation for transverse sections of a channel has the form of a non-locally driven Burgers equation. With fixed coefficients this equation admits an asymptotic similarity solution. Ratios of the granular transport coefficients can therefore be extracted from the shape of channels that have evolved in steady driving conditions.
Flow of Antarctic Bottom Water at the output of the Vema Channel
NASA Astrophysics Data System (ADS)
Tarakanov, R. Yu.; Morozov, E. G.
The pathways of the coldest part of the Antarctic Bottom Water (AABW) with potential temperature ? < 0.0░C in the Vema Channel and the pathways of this water flowing out of the channel to the Brazil Basin are studied on the basis of the data collected during the Russian expeditions in 2003, 2009-2012, and historical CTD data. It is shown that the AABW flows to the north in the Vema Channel as two streams, one of which is located in the deep channel and the other approximately 300 m higher over the western slope of the channel. It was found that the northern end of the deep channel is not located near 26░40' S, 34░00' W as a widening in the northern direction to the Brazil Basin as follows from the dataset of digital topography (Smith and Sandwell, 1997) but continues in the eastern direction. A weaker northerly flow of the cold AABW was also found from the results of the measurements in 2012, which is confined to a branch of the Vema Channel continuation.
A high-frequency warm shallow water acoustic communications channel model and measurements.
Chitre, Mandar
Underwater acoustic communication is a core enabling technology with applications in ocean monitoring using remote sensors and autonomous underwater vehicles. One of the more challenging underwater acoustic communication channels is the medium-range very shallow warm-water channel, common in tropical coastal regions. This channel exhibits two key features-extensive time-varying multipath and high levels of non-Gaussian ambient noise due to snapping shrimp-both of which limit the performance of traditional communication techniques. A good understanding of the communications channel is key to the design of communication systems. It aids in the development of signal processing techniques as well as in the testing of the techniques via simulation. In this article, a physics-based channel model for the very shallow warm-water acoustic channel at high frequencies is developed, which are of interest to medium-range communication system developers. The model is based on ray acoustics and includes time-varying statistical effects as well as non-Gaussian ambient noise statistics observed during channel studies. The model is calibrated and its accuracy validated using measurements made at sea. PMID:18189549
Intracellular chloride channel protein CLIC1 regulates macrophage function through modulation of phagosomal acidification
Jiang, Lele; Salao, Kanin; Li, Hui; Rybicka, Joanna M.; Yates, Robin M.; Luo, Xu Wei; Shi, Xin Xin; Kuffner, Tamara; Tsai, Vicky Wang-Wei; Husaini, Yasmin; Wu, Liyun; Brown, David A.; Grewal, Thomas; Brown, Louise J.; Curmi, Paul M. G.; Breit, Samuel N.
Summary Intracellular chloride channel protein 1 (CLIC1) is a 241 amino acid protein of the glutathione S transferase fold family with redox- and pH-dependent membrane association and chloride ion channel activity. Whilst CLIC proteins are evolutionarily conserved in Metazoa, indicating an important role, little is known about their biology. CLIC1 was first cloned on the basis of increased expression in activated macrophages. We therefore examined its subcellular localisation in murine peritoneal macrophages by immunofluorescence confocal microscopy. In resting cells, CLIC1 is observed in punctate cytoplasmic structures that do not colocalise with markers for endosomes or secretory vesicles. However, when these macrophages phagocytose serum-opsonised zymosan, CLIC1 translocates onto the phagosomal membrane. Macrophages from CLIC1?/? mice display a defect in phagosome acidification as determined by imaging live cells phagocytosing zymosan tagged with the pH-sensitive fluorophore Oregon Green. This altered phagosomal acidification was not accompanied by a detectable impairment in phagosomal-lysosomal fusion. However, consistent with a defect in acidification, CLIC1?/? macrophages also displayed impaired phagosomal proteolytic capacity and reduced reactive oxygen species production. Further, CLIC1?/? mice were protected from development of serum transfer induced K/BxN arthritis. These data all point to an important role for CLIC1 in regulating macrophage function through its ion channel activity and suggest it is a suitable target for the development of anti-inflammatory drugs. PMID:22956539
Golgi Anti-apoptotic Proteins Are Highly Conserved Ion Channels That Affect Apoptosis and Cell Migration*
Carrara, Guia; Saraiva, Nuno; Parsons, Maddy; Byrne, Bernadette; Prole, David L.; Taylor, Colin W.; Smith, Geoffrey L.
Golgi anti-apoptotic proteins (GAAPs) are multitransmembrane proteins that are expressed in the Golgi apparatus and are able to homo-oligomerize. They are highly conserved throughout eukaryotes and are present in some prokaryotes and orthopoxviruses. Within eukaryotes, GAAPs regulate the Ca2+ content of intracellular stores, inhibit apoptosis, and promote cell adhesion and migration. Data presented here demonstrate that purified viral GAAPs (vGAAPs) and human Bax inhibitor 1 form ion channels and that vGAAP from camelpox virus is selective for cations. Mutagenesis of vGAAP, including some residues conserved in the recently solved structure of a related bacterial protein, BsYetJ, altered the conductance (E207Q and D219N) and ion selectivity (E207Q) of the channel. Mutation of residue Glu-207 or -178 reduced the effects of GAAP on cell migration and adhesion without affecting protection from apoptosis. In contrast, mutation of Asp-219 abrogated the anti-apoptotic activity of GAAP but not its effects on cell migration and adhesion. These results demonstrate that GAAPs are ion channels and define residues that contribute to the ion-conducting pore and affect apoptosis, cell adhesion, and migration independently. PMID:25713081
Increasing mutagenicity of SŃo Gonńalo Channel waters based on the Allium cepa test.
Paiva, T S; Garcias, G L; Martino-Roth, M G
The SŃo Gonńalo Channel is of great importance to the conservation of local biodiversity; it also is a water supply source of the city of Pelotas, Brazil, and the surrounding region. We examined the mutagenic activity of its waters. The following items were seasonally investigated in Allium cepa root radicular meristem cells: mitotic index, mitotic anomalies, interphase anomalies, and total anomalies. Water samples were collected from four different stations, Lock Dam, Santa Bßrbara Channel, Pelotas Creek, and Barra do Laranjal. A drinking water negative control was used. For each sampling station, 8000 cells were counted, 2000 of which by repetition. The data were computed on a database (SPSS), and then analyzed by the chi-square test and the Mann-Whitney U-test. In 2005, the channel water provoked a significantly greater number of anomalies than the control water. The number of anomalies increased in 2007. This suggests that there was an increase in toxic substances in the channel over the years. PMID:19291879
Direct Detection of Membrane Channels from Gels Using Water-in-Oil Droplet Bilayers
Wallace, Mark
arrhythmia, periodic paralysis, epilepsy, and diabetes.1-3 Protein pores are nonspecific channels that pass other medicines for unwanted side effects.2,10 Advances in these areas require much higher throughput in many situations. Alternative emulsion-based approaches to forming bilayers have also been reported,15
Simulations of the Effects of Water Vapor, Cloud Liquid Water, and Ice on AMSU Moisture Channel Brightness Temperatures
Bradley M. Muller; Henry E. Fuelberg; Xuwu Xiang
Radiative transfer simulations are performed to determine how water vapor and nonprecipitating cloud liquid water and ice particles within typical midlatitude atmospheres affect brightness temperatures TB's of moisture sounding channels used in the Advanced Microwave Sounding Unit (AMSU) and AMSU-like instruments. The purpose is to promote a general understanding of passive top-of-atmosphere TB's for window frequencies at 23.8, 89.0, and
Basolateral targeting and microtubule-dependent transcytosis of the aquaporin-2 water channel.
Yui, Naofumi; Lu, Hua A J; Chen, Ying; Nomura, Naohiro; Bouley, Richard; Brown, Dennis
The aquaporin-2 (AQP2) water channel relocates mainly to the apical plasma membrane of collecting duct principal cells after vasopressin (VP) stimulation. AQP2 transport to this membrane domain is assumed to be a direct route involving recycling of intracellular vesicles. However, basolateral plasma membrane expression of AQP2 is observed in vivo in principal cells. Here, we asked whether there is a transcytotic pathway of AQP2 trafficking between apical and basolateral membranes. We used MDCK cells in which AQP2 normally accumulates apically after VP exposure. In contrast, both site-specific biotinylation and immunofluorescence showed that AQP2 is strongly accumulated in the basolateral membrane, along with the endocytic protein clathrin, after a brief cold shock (4░C). This suggests that AQP2 may be constitutively targeted to basolateral membranes and then retrieved by clathrin-mediated endocytosis at physiological temperatures. Rab11 does not accumulate in basolateral membranes after cold shock, suggesting that the AQP2 in this location is not associated with Rab11-positive vesicles. After rewarming (37░C), basolateral AQP2 staining is diminished and it subsequently accumulates at the apical membrane in the presence of VP/forskolin, suggesting that transcytosis can be followed by apical insertion of AQP2. This process is inhibited by treatment with colchicine. Our data suggest that the cold shock procedure reveals the presence of microtubule-dependent AQP2 transcytosis, which represents an indirect pathway of apical AQP2 delivery in these cells. Furthermore, our data indicate that protein polarity data obtained from biotinylation assays, which require cells to be cooled to 4░C during the labeling procedure, should be interpreted with caution. PMID:23015545
Enantioselective Protein-Sterol Interactions Mediate Regulation of Both Prokaryotic and Eukaryotic Inward Rectifier K+ Channels by Cholesterol
D'Avanzo, Nazzareno; Hyrc, Krzysztof; Enkvetchakul, Decha; Covey, Douglas F.; Nichols, Colin G.
Cholesterol is the major sterol component of all mammalian cell plasma membranes and plays a critical role in cell function and growth. Previous studies have shown that cholesterol inhibits inward rectifier K+ (Kir) channels, but have not distinguished whether this is due directly to protein-sterol interactions or indirectly to changes in the physical properties of the lipid bilayer. Using purified bacterial and eukaryotic Kir channels reconstituted into liposomes of controlled lipid composition, we demonstrate by 86Rb+ influx assays that bacterial Kir channels (KirBac1.1 and KirBac3.1) and human Kir2.1 are all inhibited by cholesterol, most likely by locking the channels into prolonged closed states, whereas the enantiomer, ent-cholesterol, does not inhibit these channels. These data indicate that cholesterol regulates Kir channels through direct protein-sterol interactions likely taking advantage of an evolutionarily conserved binding pocket. PMID:21559361
Effect of Water Content on Glass Transition and Protein Aggregation of Whey Protein Powders During Short-Term Storage
Peng Zhou; Theodore P. Labuza
The objectives of this study were to investigate the moisture-induced protein aggregation of whey protein powders and to elucidate\\u000a the relationship of protein stability with respect to water content and glass transition. Three whey protein powder types\\u000a were studied: whey protein isolate (WPI), whey protein hydrolysates (WPH), and beta-lactoglobulin (BLG). The water sorption\\u000a isotherms were determined at 23 and 45░C,
Unstructured to structured transition of an intrinsically disordered protein peptide in coupling Ca▓?-sensing and SK channel activation.
Zhang, Miao; Pascal, John M; Zhang, Ji-Fang
Most proteins, such as ion channels, form well-organized 3D structures to carry out their specific functions. A typical voltage-gated potassium channel subunit has six transmembrane segments (S1-S6) to form the voltage-sensing domain and the pore domain. Conformational changes of these domains result in opening of the channel pore. Intrinsically disordered (ID) proteins/peptides are considered equally important for the protein functions. However, it is difficult to explore the structural features underlying the functions of ID proteins/peptides by conventional methods, such as X-ray crystallography, because of the flexibility of their secondary structures. Unlike voltage-gated potassium channels, families of small- and intermediate-conductance Ca(2+)-activated potassium (SK/IK) channels with important roles in regulating membrane excitability are activated exclusively by Ca(2+)-bound calmodulin (CaM). Upon binding of Ca(2+) to CaM, a 2 ū 2 structure forms between CaM and the CaM-binding domain. A channel fragment that connects S6 and the CaM-binding domain is not visible in the protein crystal structure, suggesting that this fragment is an ID fragment. Here we show that the conformation of the ID fragment in SK channels becomes readily identifiable in the presence of NS309, the most potent compound that potentiates the channel activities. This well-defined conformation of the ID fragment, stabilized by NS309, increases the channel open probability at a given Ca(2+) concentration. Our results demonstrate that the ID fragment, itself a target for drugs modulating SK channel activities, plays a unique role in coupling Ca(2+) sensing by CaM and mechanical opening of SK channels. PMID:23487779
Evaluation of coastal Bermuda grass protein extract as a substitute for fishmeal in practical diets for channel catfish (Ictalurus punctatus)
Buentello, J. Alejandro
In response to concerns over availability and cost of fishmeal for aquaculture feeds, a study was conducted to evaluate the suitability of a protein extract from coastal Bermuda grass for channel catfish (Ictalurus punctatus). The coastal Bermuda...
Evaluation of coastal Bermuda grass protein extract as a substitute for fishmeal in practical diets for channel catfish (Ictalurus punctatus)Ā
Buentello, J. Alejandro
In response to concerns over availability and cost of fishmeal for aquaculture feeds, a study was conducted to evaluate the suitability of a protein extract from coastal Bermuda grass for channel catfish (Ictalurus punctatus). The coastal Bermuda...
Protein-spanning water networks and implications for prediction of protein-protein interactions mediated through hydrophobic effects.
Cui, Di; Ou, Shuching; Patel, Sandeep
Hydrophobic effects, often conflated with hydrophobic forces, are implicated as major determinants in biological association and self-assembly processes. Protein-protein interactions involved in signaling pathways in living systems are a prime example where hydrophobic effects have profound implications. In the context of protein-protein interactions, a priori knowledge of relevant binding interfaces (i.e., clusters of residues involved directly with binding interactions) is difficult. In the case of hydrophobically mediated interactions, use of hydropathy-based methods relying on single residue hydrophobicity properties are routinely and widely used to predict propensities for such residues to be present in hydrophobic interfaces. However, recent studies suggest that consideration of hydrophobicity for single residues on a protein surface require accounting of the local environment dictated by neighboring residues and local water. In this study, we use a method derived from percolation theory to evaluate spanning water networks in the first hydration shells of a series of small proteins. We use residue-based water density and single-linkage clustering methods to predict hydrophobic regions of proteins; these regions are putatively involved in binding interactions. We find that this simple method is able to predict with sufficient accuracy and coverage the binding interface residues of a series of proteins. The approach is competitive with automated servers. The results of this study highlight the importance of accounting of local environment in determining the hydrophobic nature of individual residues on protein surfaces. PMID:25204743
Differential regulation of G protein-gated inwardly rectifying K+ channel kinetics by distinct domains of RGS8
Jeong, Seong-Woo; Ikeda, Stephen R
The contribution of endogenous regulators of G protein signalling (RGS) proteins to G protein modulated inwardly rectifying K+ channel (GIRK) activation/deactivation was examined by expressing mutants of G?oA insensitive to both pertussis toxin (PTX) and RGS proteins in rat sympathetic neurons. GIRK channel modulation was reconstituted in PTX-treated rat sympathetic neurons following heterologous expression of G protein subunits. Under these conditions, noradrenaline-evoked GIRK channel currents displayed: (1) a prominent lag phase preceding activation, (2) retarded activation and deactivation kinetics, and (3) a lack of acute desensitization. Unexpectedly, heterologous expression of RGS8 in neurons expressing PTX-i-RGS-insensitive G?oA shortened the lag phase and restored rapid activation, but retarded the deactivation phase further. These effects were found to arise from the N-terminus, but not the core domain, of RGS8 thus suggesting actions on channel modulation independently of GTPase acceleration. These findings indicate that different domains of RGS8 make distinct contributions to the temporal regulation of GIRK channels. The RGS8 core domain accelerates termination of the G-protein cycle presumably by increasing G? GTPase activity. In contrast, the N-terminal domain of RGS8 appears to promote entry into the G protein cycle, possibly by enhancing coupling of receptors to the G protein heterotrimer. Together, these opposing effects should allow for an increase in temporal fidelity without a dramatic decrease in signal strength. PMID:11533127
Thermodynamic study of protein phases formation and clustering in model water-protein-salt solutions.
Rozhkov, Sergey P; Goryunov, Andrey S
Thermodynamic analysis of the water-protein-salt system, based on the description of the spinodal curve, has been carried out in various coordinate systems: (water chemical potential, protein concentration m(2)); (protein "solubility" log S, salt concentration m(3)); (effective temperature, critical composition of the system m(2)/m(3)). Such presentations explain the existence of diagrams with normal and retrograde protein solubility as a result of straightforward effect of ions present in solution as well as some features of the widely used phase diagram in coordinates (temperature, protein concentration). Analytic expressions for coefficients K and b of the salting out equation log S=-K.m(3)+b as functions of protein charge and protein adsorbed ions have been obtained and identified with the spinodal characteristic points reflecting quasi-equilibrium between protein-lean phase and dense protein-rich phase. Liquid-liquid, liquid-solid phase transitions, dynamic protein clusters and second virial coefficient that characterize interaction between solution components have been thus interrelated. The results of our thermodynamic analysis have been compared with the data reported for lysozyme . PMID:20494508
Studies on protein transmission in thin channel flow module: the role of dean vortices for improving mass transfer
Jasmedh Kaur; G. P Agarwal
This study is mainly comprised of evaluating mass transfer coefficient from the volumetric flux and protein transmission data in a thin channel flow module. Dilute solutions of lysozyme were ultrafiltered through hydrophilic membrane (cellulose acetate type) with molecular weight cut off (MWCO) of 30,000. Experiments were performed in thin channel flow (TCF10) configuration and data for transmission and volumetric flux
Adaptive resolution simulation of an atomistic protein in MARTINI water
Zavadlav, Julija [Laboratory for Molecular Modeling, National Institute of Chemistry, Hajdrihova 19, SI-1001 Ljubljana (Slovenia)] [Laboratory for Molecular Modeling, National Institute of Chemistry, Hajdrihova 19, SI-1001 Ljubljana (Slovenia); Melo, Manuel Nuno; Marrink, Siewert J., E-mail: email@example.com [Groningen Biomolecular Sciences and Biotechnology Institute and Zernike Institute for Advanced Materials, University of Groningen, Nijenborgh 7, 9747 AG Groningen (Netherlands); Praprotnik, Matej, E-mail: email@example.com [Laboratory for Molecular Modeling, National Institute of Chemistry, Hajdrihova 19, SI-1001 Ljubljana (Slovenia) [Laboratory for Molecular Modeling, National Institute of Chemistry, Hajdrihova 19, SI-1001 Ljubljana (Slovenia); Department of Physics, Faculty of Mathematics and Physics, University of Ljubljana, Jadranska 19, SI-1000 Ljubljana (Slovenia)
We present an adaptive resolution simulation of protein G in multiscale water. We couple atomistic water around the protein with mesoscopic water, where four water molecules are represented with one coarse-grained bead, farther away. We circumvent the difficulties that arise from coupling to the coarse-grained model via a 4-to-1 molecule coarse-grain mapping by using bundled water models, i.e., we restrict the relative movement of water molecules that are mapped to the same coarse-grained bead employing harmonic springs. The water molecules change their resolution from four molecules to one coarse-grained particle and vice versa adaptively on-the-fly. Having performed 15?ns long molecular dynamics simulations, we observe within our error bars no differences between structural (e.g., root-mean-squared deviation and fluctuations of backbone atoms, radius of gyration, the stability of native contacts and secondary structure, and the solvent accessible surface area) and dynamical properties of the protein in the adaptive resolution approach compared to the fully atomistically solvated model. Our multiscale model is compatible with the widely used MARTINI force field and will therefore significantly enhance the scope of biomolecular simulations.
The Proapoptotic Influenza A Virus Protein PB1-F2 Forms a Nonselective Ion Channel
Henkel, Michael; Mitzner, David; Henklein, Peter; Meyer-Almes, Franz-Josef; Moroni, Anna; DiFrancesco, Mattia L.; Henkes, Leonhard M.; Kreim, Michael; Kast, Stefan M.; Schubert, Ulrich; Thiel, Gerhard
Background PB1-F2 is a proapoptotic influenza A virus protein of approximately 90 amino acids in length that is located in the nucleus, cytosol and in the mitochondria membrane of infected cells. Previous studies indicated that the molecule destabilizes planar lipid bilayers and has a strong inherent tendency for multimerization. This may be correlate with its capacity to induce mitochondrial membrane depolarization. Methodology/Principal Findings Here, we investigated whether PB1-F2 is able to form ion channels within planar lipid bilayers and microsomes. For that purpose, a set of biologically active synthetic versions of PB1-F2 (sPB1-F2) derived from the IAV isolates A/Puerto Rico/8/34(H1N1) (IAVPR8), from A/Brevig Mission/1/1918(H1N1) (IAVSF2) or the H5N1 consensus sequence (IAVBF2) were used. Electrical and fluorimetric measurements show that all three peptides generate in planar lipid bilayers or in liposomes, respectively, a barely selective conductance that is associated with stochastic channel type fluctuations between a closed state and at least two defined open states. Unitary channel fluctuations were also generated when a truncated protein comprising only the 37 c-terminal amino acids of sPB1-F2 was reconstituted in bilayers. Experiments were complemented by extensive molecular dynamics simulations of the truncated fragment in a lipid bilayer. The results indicate that the c-terminal region exhibits a slightly bent helical fold, which is stable and remains embedded in the bilayer for over 180 ns. Conclusion/Significance The data support the idea that PB1-F2 is able to form protein channel pores with no appreciable selectivity in membranes and that the c-terminus is important for this function. This information could be important for drug development. PMID:20559552
4D photogrammetric technique to study free surface water in open channels
NASA Astrophysics Data System (ADS)
Aubķ, Damien; Berkaoui, Amine; Vinatier, Fabrice; Bailly, Jean-Stķphane; Belaud, Gilles
Characteristics of three-dimensional surface water are considered as the most valuable information to understand hydrodynamic phenomena in open channel flow. An accurate and coherent description of the free water surface morphology improves the accuracy of hydraulic models which study river processes. However, amongst existing techniques to measure three-dimensional surface, stereo-photogrammetry is clearly the most effective technique to obtain an instantaneous and high accurate 3D free water surface and it's suitable to both flume and field condition. Our study aims at developing this technique in two controlled channels, one in interior with glass borders (length: 6 m, width: 0.3 m and depth: 0.5 m) and one outside with cement borders (length: 13 m, width: 0.7 m and depth: 0.4 m). A system consisting in three NIKON-D3200 cameras, mounted to an adjustable tripod head, which is fixed to an inverted aluminium T-bar with the center camera higher than the two side cameras. Each camera is fitted with a 28 mm lens and cameras are synchronized using a Phottix(R) system. The system was mounted at a downstream position from the channel with an oblique configuration. A series of pictures taken at a 3 s interval during the water weight bearing were reported and analyzed using the Photoscan Pro(R) software for image matching. Validation procedure of the technique was realized using an orthophotography of the lateral border of the interior channel to delimit the line of water surface, and using a video capture of a slide fixed inside the outside channel. A high resolution and dynamic elevation map of the surface water was constructed. Our study give encouraging results, with a good capture of water surface morphology and a limited occlusion issues. The confrontation of the results with the validation dataset highlight limitations that need to be discussed with the audience.
Estimating Small Scale River Channel Roughness Using a Through-water Photo-based technique
NASA Astrophysics Data System (ADS)
James, M. R.; Scarborough, F.; Folkard, A. M.
Channel roughness is critical to the understanding of fluvial geomorphology and hydrology due to its connection with the transportation of sediment and effect on flow discharge. Due to manual measurement methods being costly and time consuming, and traditional visual observation methods being subjective, we have explored the use of a close-range remote sensing approach, based on through-water photography to estimate channel characteristics. Previous similar photo-based measurements have focused on estimating water depth by correcting data from stereo image pairs for refraction at the water surface. Here, we extend this approach to multi-image data sets, and implement refraction correction for data from commonly used 'structure from motion' based software. The accuracy of applied corrections is assessed in a laboratory setting using a gravel surface submerged at a range of water depths. We demonstrate the approach in the field by photographing cross sections to produce high density point clouds and hence digital elevation models of the stream bed. Correcting submerged regions for refraction effects allows channel characteristics such as wetted perimeter and water depth to be estimated. We explore the use of parameters thus estimated for deriving coefficients of channel flow resistance such as Manning's coefficient.
Hydration-dependent dynamic crossover phenomenon in protein hydration water
NASA Astrophysics Data System (ADS)
Wang, Zhe; Fratini, Emiliano; Li, Mingda; Le, Peisi; Mamontov, Eugene; Baglioni, Piero; Chen, Sow-Hsin
The characteristic relaxation time ? of protein hydration water exhibits a strong hydration level h dependence. The dynamic crossover is observed when h is higher than the monolayer hydration level hc=0.2-0.25 and becomes more visible as h increases. When h is lower than hc, ? only exhibits Arrhenius behavior in the measured temperature range. The activation energy of the Arrhenius behavior is insensitive to h, indicating a local-like motion. Moreover, the h dependence of the crossover temperature shows that the protein dynamic transition is not directly or solely induced by the dynamic crossover in the hydration water.
An investigation of channel flow with a smooth air-water interface
NASA Astrophysics Data System (ADS)
Madad, Reza; Elsnab, John; Chin, Cheng; Klewicki, Joseph; Marusic, Ivan
Experiments and numerical simulation are used to investigate fully developed laminar and turbulent channel flow with an air-water interface as the lower boundary condition. Laser Doppler velocimetry measurements of streamwise and wall-normal velocity components are made over a range of Reynolds number based upon channel height and bulk velocity from 1100 to 4300, which encompasses the laminar, transitional and low Reynolds numbers turbulent regimes. The results show that the airflow statistics near the stationary wall are not significantly altered by the air-water moving interface and reflect those found in channel flows. The mean statistics on the water interface side largely exhibit results similar to simulated Poiseuille-Couette flow (PCF) with a solid moving wall. For second-order statistics, however, the simulation and experimental results show some discrepancies near the moving water surface, suggesting that a full two-phase simulation is required. A momentum and energy transport tubes analysis is investigated for laminar and turbulent PCFs. This analysis builds upon the classical notion of a streamtube and indicates that part of the energy from the pressure gradient is transported towards the stationary wall and is dissipated as heat inside the energy tubes, while the remainder is transmitted to the moving wall. For the experiments, the airflow energy is transmitted towards the water to overcome the drag force and drive the water forward; therefore, the amount of energy transferred to the water is higher than the energy transferred to a solid moving wall.
2D IR spectroscopy reveals the role of water in the binding of channel-blocking drugs to the influenza M2 channel
NASA Astrophysics Data System (ADS)
Ghosh, Ayanjeet; Wang, Jun; Moroz, Yurii S.; Korendovych, Ivan V.; Zanni, Martin; DeGrado, William F.; Gai, Feng; Hochstrasser, Robin M.
Water is an integral part of the homotetrameric M2 proton channel of the influenza A virus, which not only assists proton conduction but could also play an important role in stabilizing channel-blocking drugs. Herein, we employ two dimensional infrared (2D IR) spectroscopy and site-specific IR probes, i.e., the amide I bands arising from isotopically labeled Ala30 and Gly34 residues, to probe how binding of either rimantadine or 7,7-spiran amine affects the water dynamics inside the M2 channel. Our results show, at neutral pH where the channel is non-conducting, that drug binding leads to a significant increase in the mobility of the channel water. A similar trend is also observed at pH 5.0 although the difference becomes smaller. Taken together, these results indicate that the channel water facilitates drug binding by increasing its entropy. Furthermore, the 2D IR spectral signatures obtained for both probes under different conditions collectively support a binding mechanism whereby amantadine-like drugs dock in the channel with their ammonium moiety pointing toward the histidine residues and interacting with a nearby water cluster, as predicted by molecular dynamics simulations. We believe these findings have important implications for designing new anti-influenza drugs.
Quantifying the Role of Water in Protein¢Carbohydrate Interactions
Tschampel, Sarah M.; Woods, Robert J.
Water-mediated interactions play a key role in carbohydrate¢lectin binding, where the interactions involve a conserved water that is separated from the bulk solvent and present a bridge between the side chains of the protein and the carbohydrate ligand. To apply quantum mechanical methods to examine the role of conserved waters, we present an analysis in which the relevant carbohydrate atoms are modeled by methanol, and in which the protein is replaced by a limited number of amino acid side chains. Clusters containing a conserved water and a representative amino acid fragment were also examined to determine the influence of amino acid side chains on interaction energies. To quantify the differential binding energies of methanol versus water, quantum mechanical calculations were performed at the B3LYP/6-311++G(3df,3pd)//B3LYP/6-31+G(d) level in which either a methanol molecule was bound to the conserved water (liganded state) or in which a water molecule replaces the methanol (unliganded state). Not surprisingly, the binding of a water to clusters containing charged amino acid side chains was more favorable by 1.55 to 7.23 kcal/mol than that for the binding of a water to the corresponding pure water clusters. In contrast, the binding energy of water to clusters containing polar-uncharged amino acid side chains ranged from 4.35 kcal/mol less favorable to 4.72 kcal/mol more favorable than for binding to the analogous pure water clusters. The overall trend for the binding of methanol versus water, in any of the clusters, favored methanol by an average value of 1.05 kcal/mol. To extend these studies to a complex between a protein (Concanavalin A) and its carbohydrate ligand, a cluster was examined that contained the side chains of three key amino acids, namely asparagine, aspartate, and arginine, as well as a key water molecule, arranged as in the X-ray diffraction structure of Con A. Again, using methanol as a model for the endogenous carbohydrate ligand, energies of ?5.94 kcal/mol and ?5.70 kcal/mol were obtained for the binding of methanol and water, respectively, to the Con A¢water cluster. The extent to which cooperativity enhanced the binding energies has been quantified in terms of nonadditive three-body contributions. In general, the binding of water or methanol to neutral dimers formed cooperative clusters; in contrast, the cooperativity in charged clusters depended on the overall geometry as well as the charge. PMID:16906231
The experimental study of the features of water flowing through a sharpcrested weir in channel
NASA Astrophysics Data System (ADS)
Turalina, Dinara; Yembergenova, Dinara; Alibayeva, Karlygash
This article contains the experimental research of water flowing through a weir in a rectangular open channel. Nine regimes of water flowrate investigated in this study. Upstream water levels partially determined for each regime. The coefficient of discharge to the weir determined from the flowrate equation. The determined coefficient of discharge value compared to the value from the Rehbock formula. The diagram of values shows the dependence of the coefficient of discharge Cd on the upstream water level yc/h. Experimental study conducted on the Armfield S16 hydraulic flow demonstrator and hydraulic bench F1-10.
Generation, Comparison, and Merging of Pathways between Protein Conformations: Gating in K-Channels
Enosh, Angela; Raveh, Barak; Furman-Schueler, Ora; Halperin, Dan; Ben-Tal, Nir
We present a general framework for the generation, alignment, comparison, and hybridization of motion pathways between two known protein conformations. The framework, which is rooted in probabilistic motion-planning techniques in robotics, allows for the efficient generation of collision-free motion pathways, while considering a wide range of degrees of freedom involved in the motion. Within the framework, we provide the means to hybridize pathways, thus producing, the motion pathway of the lowest energy barrier out of the many pathways proposed by our algorithm. This method for comparing and hybridizing pathways is modular, and may be used within the context of molecular dynamics and Monte Carlo simulations. The framework was implemented within the Rosetta software suite, where the protein is represented in atomic detail. The K-channels switch between open and closed conformations, and we used the overall framework to investigate this transition. Our analysis suggests that channel-opening may follow a three-phase pathway. First, the channel unlocks itself from the closed state; second, it opens; and third, it locks itself in the open conformation. A movie that depicts the proposed pathway is available in the Supplementary Material (Movie S1) and at http://www.cs.tau.ac.il/?angela/SuppKcsA.html. PMID:18621834
Slip effects on mixed convective peristaltic transport of copper-water nanofluid in an inclined channel.
Abbasi, Fahad Munir; Hayat, Tasawar; Ahmad, Bashir; Chen, Guo-Qian
Peristaltic transport of copper-water nanofluid in an inclined channel is reported in the presence of mixed convection. Both velocity and thermal slip conditions are considered. Mathematical modelling has been carried out using the long wavelength and low Reynolds number approximations. Resulting coupled system of equations is solved numerically. Quantities of interest are analyzed through graphs. Numerical values of heat transfer rate at the wall for different parameters are obtained and examined. Results showed that addition of copper nanoparticles reduces the pressure gradient, axial velocity at the center of channel, trapping and temperature. Velocity slip parameter has a decreasing effect on the velocity near the center of channel. Temperature of nanofluid increases with increase in the Grashoff number and channel inclination angle. It is further concluded that the heat transfer rate at the wall increases considerably in the presence of copper nanoparticles. PMID:25170908
Slip Effects on Mixed Convective Peristaltic Transport of Copper-Water Nanofluid in an Inclined Channel
Abbasi, Fahad Munir; Hayat, Tasawar; Ahmad, Bashir; Chen, Guo-Qian
Peristaltic transport of copper-water nanofluid in an inclined channel is reported in the presence of mixed convection. Both velocity and thermal slip conditions are considered. Mathematical modelling has been carried out using the long wavelength and low Reynolds number approximations. Resulting coupled system of equations is solved numerically. Quantities of interest are analyzed through graphs. Numerical values of heat transfer rate at the wall for different parameters are obtained and examined. Results showed that addition of copper nanoparticles reduces the pressure gradient, axial velocity at the center of channel, trapping and temperature. Velocity slip parameter has a decreasing effect on the velocity near the center of channel. Temperature of nanofluid increases with increase in the Grashoff number and channel inclination angle. It is further concluded that the heat transfer rate at the wall increases considerably in the presence of copper nanoparticles. PMID:25170908
Electrophysiological Studies in Xenopus Oocytes for the Opening of Escherichia coli SecA-Dependent Protein-Conducting Channels
Lin, Bor-Ruei; Gierasch, Lila M.; Jiang, Chun; Tai, Phang C.
Protein translocation in Escherichia coli requires protein-conducting channels in cytoplasmic membranes to allow precursor peptides to pass through with adenosine triphosphate (ATP) hydrolysis. Here, we report a novel, sensitive method that detects the opening of the SecA-dependent protein-conducting channels at the nanogram level. E. coli inverted membrane vesicles were injected into Xenopus oocytes, and ionic currents were recorded using the two-electrode voltage clamp. Currents were observed only in the presence of E. coli SecA in conjunction with E. coli membranes. Observed currents showed outward rectification in the presence of KCl as permeable ions and were significantly enhanced by coinjection with the precursor protein proOmpA or active LamB signal peptide. Channel activity was blockable with sodium azide or adenylyl 5?-(?,?-methylene)- diphosphonate, a nonhydrolyzable ATP analogue, both of which are known to inhibit SecA protein activity. Endogenous oocyte precursor proteins also stimulated ion current activity and can be inhibited by puromycin. In the presence of puromycin, exogenous proOmpA or LamB signal peptides continued to enhance ionic currents. Thus, the requirement of signal peptides and ATP hydrolysis for the SecA-dependent currents resembles biochemical protein translocation assay with E. coli membrane vesicles, indicating that the Xenopus oocyte system provides a sensitive assay to study the role of Sec and precursor proteins in the formation of protein-conducting channels using electrophysiological methods. PMID:17530158
On the Modeling of Channel-to-Channel Oscillations in Boiling Water Reactors
Sebastien Roubaud; Michael Z. Podowski
Several modes of flow-induced oscillations have been observed in two-phase flow systems in general, and Boiling Water Nuclear Reactors (BWR) in particular. The challenges specifically associated with developing reliable predictive capabilities for possible instabilities in BWR systems are concerned with issues such as: proper identification of the dominant mode of instabilities, and the modeling of coupling between the thermal-hydraulic phenomena
Resurrecting Abandoned Proteins with Pure Water: CD and NMR Studies of Protein Fragments Solubilized in Salt-Free Water
Minfen Li; Jingxian Liu; Xiaoyuan Ran; Miaoqing Fang; Jiahai Shi; Haina Qin; June-Mui Goh; Jianxing Song
Many proteins expressed in Escherichia coli cells form inclusion bodies that are neither refoldable nor soluble in buffers. Very surprisingly, we recently discovered that all 11 buffer-insoluble protein fragments\\/domains we have, with a great diversity of cellular function, location, and molecular size, could be easily solubilized in salt-free water. The circular dichroism (CD) and NMR characterization led to classification of
Induction of aquaporin-4 water channel mRNA after focal cerebral ischemia in rat
Masaaki Taniguchi; Toshihide Yamashita; Eiji Kumura; Michio Tamatani; Akihiro Kobayashi; Takashi Yokawa; Motohiko Maruno; Amami Kato; Takanori Ohnishi; Eiji Kohmura; Masaya Tohyama; Toshiki Yoshimine
Aquaporin-4 (AQP4) is a member of a water-selective channel aquaporin-family and mainly expressed in the several structures of the brain and in the collecting duct of the kidney. Here we show its functional involvement in the water homeostasis of the ischemic brain. The expression of AQP4¢mRNA is increased in the peri-infarcted cortex during the observation period (?7 days) after MCA-occlusion,
Biogas production from water hyacinth and channel grass used for phytoremediation of industrial effluents
V Singhal; J. P. N Rai
The paper reports on the biogas production from water hyacinth (Eichhornia crassipes) and channel grass (Vallisneria spiralis) employed separately for phytoremediation of lignin and metal-rich pulp and paper mill and highly acidic distillery effluents. These plants eventually grow well in diluted effluent up to 40% (i.e., 2.5-times dilution with deionized water) and often take up metals and toxic materials from
RING finger protein 121 facilitates the degradation and membrane localization of voltage-gated sodium channels.
Ogino, Kazutoyo; Low, Sean E; Yamada, Kenta; Saint-Amant, Louis; Zhou, Weibin; Muto, Akira; Asakawa, Kazuhide; Nakai, Junichi; Kawakami, Koichi; Kuwada, John Y; Hirata, Hiromi
Following their synthesis in the endoplasmic reticulum (ER), voltage-gated sodium channels (NaV) are transported to the membranes of excitable cells, where they often cluster, such as at the axon initial segment of neurons. Although the mechanisms by which NaV channels form and maintain clusters have been extensively examined, the processes that govern their transport and degradation have received less attention. Our entry into the study of these processes began with the isolation of a new allele of the zebrafish mutant alligator, which we found to be caused by mutations in the gene encoding really interesting new gene (RING) finger protein 121 (RNF121), an E3-ubiquitin ligase present in the ER and cis-Golgi compartments. Here we demonstrate that RNF121 facilitates two opposing fates of NaV channels: (i) ubiquitin-mediated proteasome degradation and (ii) membrane localization when coexpressed with auxiliary NaV? subunits. Collectively, these results indicate that RNF121 participates in the quality control of NaV channels during their synthesis and subsequent transport to the membrane. PMID:25691753
The p7 protein of hepatitis C virus forms structurally plastic, minimalist ion channels.
Chandler, Danielle E; Penin, Franńois; Schulten, Klaus; Chipot, Christophe
Hepatitis C virus (HCV) p7 is a membrane-associated oligomeric protein harboring ion channel activity. It is essential for effective assembly and release of infectious HCV particles and an attractive target for antiviral intervention. Yet, the self-assembly and molecular mechanism of p7 ion channelling are currently only partially understood. Using molecular dynamics simulations (aggregate time 1.2 Ąs), we show that p7 can form stable oligomers of four to seven subunits, with a bias towards six or seven subunits, and suggest that p7 self-assembles in a sequential manner, with tetrameric and pentameric complexes forming as intermediate states leading to the final hexameric or heptameric assembly. We describe a model of a hexameric p7 complex, which forms a transiently-open channel capable of conducting ions in simulation. We investigate the ability of the hexameric model to flexibly rearrange to adapt to the local lipid environment, and demonstrate how this model can be reconciled with low-resolution electron microscopy data. In the light of these results, a view of p7 oligomerization is proposed, wherein hexameric and heptameric complexes may coexist, forming minimalist, yet robust functional ion channels. In the absence of a high-resolution p7 structure, the models presented in this paper can prove valuable as a substitute structure in future studies of p7 function, or in the search for p7-inhibiting drugs. PMID:23028296
Integration of thermal and osmotic regulation of water homeostasis: the role of TRPV channels.
Sladek, Celia D; Johnson, Alan Kim
Maintenance of body water homeostasis is critical for preventing hyperthermia, because evaporative cooling is the most efficient means of dissipating excess body heat. Water homeostasis is achieved by regulation of water intake and water loss by the kidneys. The former is achieved by sensations of thirst that motivate water acquisition, whereas the latter is regulated by the antidiuretic action of vasopressin. Vasopressin secretion and thirst are stimulated by increases in the osmolality of the extracellular fluid as well as decreases in blood pressure and/or blood volume, signals that are precipitated by water depletion associated with the excess evaporative water loss required to prevent hyperthermia. In addition, they are stimulated by increases in body temperature. The sites and molecular mechanisms involved in integrating thermal and osmotic regulation of thirst and vasopressin secretion are reviewed here with a focus on the role of the thermal and mechanosensitive transient receptor potential-vanilloid (TRPV) family of ion channels. PMID:23883678
Pore waters regulate ion permeation in a calcium release-activated calcium channel
Dong, Hao; Fiorin, Giacomo; Carnevale, Vincenzo; Treptow, Werner; Klein, Michael L.
The recent crystal structure of Orai, the pore unit of a calcium release-activated calcium (CRAC) channel, is used as the starting point for molecular dynamics and free-energy calculations designed to probe this channelÆs conduction properties. In free molecular dynamics simulations, cations localize preferentially at the extracellular channel entrance near the ring of Glu residues identified in the crystal structure, whereas anions localize in the basic intracellular half of the pore. To begin to understand ion permeation, the potential of mean force (PMF) was calculated for displacing a single Na+ ion along the pore of the CRAC channel. The computed PMF indicates that the central hydrophobic region provides the major hindrance for ion diffusion along the permeation pathway, thereby illustrating the nonconducting nature of the crystal structure conformation. Strikingly, further PMF calculations demonstrate that the mutation V174A decreases the free energy barrier for conduction, rendering the channel effectively open. This seemingly dramatic effect of mutating a nonpolar residue for a smaller nonpolar residue in the pore hydrophobic region suggests an important role for the latter in conduction. Indeed, our computations show that even without significant channel-gating motions, a subtle change in the number of pore waters is sufficient to reshape the local electrostatic field and modulate the energetics of conduction, a result that rationalizes recent experimental findings. The present work suggests the activation mechanism for the wild-type CRAC channel is likely regulated by the number of pore waters and hence pore hydration governs the conductance. PMID:24101457
Short Paper:R&D of a Dual mode acoustic modem testbed for shallow water channels
Kastner, Ryan
Short Paper:R&D of a Dual mode acoustic modem testbed for shallow water channels Feng TONG1, which urged the R&D of high performance underwater acoustic (UWA) modems. However, as underwater modem testbed system, which enables the switch between DSSS and OFDM scheme to accommodate different
P2X Receptors: Epithelial Ion Channels and Regulators of Salt and Water Transport
Scott S. P. Wildman; Brian F. King
When the results from electrophysiological studies of renal epithelial cells are combined with data from in vivo tubule microperfusion experiments and immunohistochemical surveys of the nephron, the accumulated evidence suggests that ATP-gated ion channels, P2X receptors, play a specialized role in the regulation of ion and water movement across the renal tubule and are integral to electrolyte and fluid homeostasis.
Inhibition of Water Channels by HgCl2 in Intact Wheat Root Cells1
Zhang, Wen-Hao; Tyerman, Stephen D.
To assess the extent of water flow through channels in the membranes of intact higher plant cells, the effects of HgCl2 on hydraulic conductivity (LP) of wheat (Triticum aestivum L.) root cells were investigated using a pressure probe. The LP of root cells was reduced by 75% in the presence of 100 ?m HgCl2. The K+-channel blocker tetraethylammonium had no effect on the LP at concentrations that normally block K+ channels. HgCl2 rapidly depolarized the membrane potential (Vm) of the root cells. The dose-response relationship of inhibition of LP and depolarization of Vm were not significantly different, with half-maximal inhibition occurring at 4.6 and 7.8 ?m, respectively. The inhibition of LP and the depolarization of Vm caused by HgCl2 were partially reversed by ?-mercaptoethanol. The inhibition of LP by HgCl2 was similar in magnitude to that caused by hypoxia, and the addition of HgCl2 to hypoxia-treated cells did not result in further inhibition. We compared the LP of intact cells with that predicted from a model of cortical cells incorporating water flow across both the plasma membrane and the tonoplast using measured values of water permeability from isolated membranes, and found that HgCl2 has other effects in addition to the direct inhibition of water channels. PMID:10398721
On the turbulent flow around water turbines placed in an open channel: an experimental study
NASA Astrophysics Data System (ADS)
Sotiropoulos, F.; Chamorro, L. P.; Arndt, R.
A growing interest in water turbines (using tidal, river, marine currents) has been observed during the last few years. Fundamental understanding of the turbulent flow around the water turbines is crucial to predict the potential effects of these structures on the local morphology, water flow and power available in the current, among others. In this study, a series of model water turbines (single and an aligned array) of 50 cm rotor diameter were placed in the main channel of the Saint Anthony Falls Laboratory at the University of Minnesota. The main channel is approx 2.5 m wide, 1.8 m height and 85 m long. Flow around the water turbines were analyzed under subcritical conditions. Turbine hub heights coincided with the channel mid height. A series of acoustic Doppler anemometers (ADV) were used to obtain 3 velocity components of the flow at a rate of 200 Hz. Selected streamwise and spanwise vertical planes were measured to describe the kinematics around the water turbines. Potential interactions with the lateral walls were also addressed. High order statistics (mean velocity, turbulence intensities and Reynolds stresses) as well as two point correlations and spectra were computed to infer fundamental differences and similitude with their counterparts, the wind turbines.
Triggering a Wet Climate on Mars: The Role of Outflow Channels in Martian Water Cycles
NASA Astrophysics Data System (ADS)
Santiago, D.; Asphaug, E. I.; Colaprete, A.
The triggering of a robust water cycle on Mars has been hypothesized to be caused by gigantic flooding events evidenced by outflow channels. Here we use the Ames Mars General Circulation Model (MGCM) to study how these presumably abrupt eruptions of water (Carr,1996) affected the climate of Mars. We model where the water ultimately went as part of a transient hydrologic cycle. Chryse Planitia, east of Tharsis, has evidence for multiple water outflow channels. One of the largest channels is Ares Valles, which was carved by floods with estimated water volumes of order 10^5 km^2 (Andrews-Hanna, 2007 & Carr, 1996). Outflow discharge rate estimates range from 10^6 to 10^7 m^3/seconds or greater (Andrews-Hanna & Phillips, 2007, Harrison & Grimm, 2008). Studies suggest that outflow channels formed with smaller, successive floods instead of a single large flood (Wilson, et al.,2004). Warner et al. (2009) suggest up to six outflow events for the formation of Ares Valles, while estimates for another large outflow, Kasei Valles, might have been flooded by over two thousand floods with a total water volume of 5.5 x 10^5 km^3 (Harrison & Grimm, 2008). By adding water to the surface of Mars at the given outflow rate, as an expanding one-layer lake, we are able to study quantitatively how these outflow events influenced Mars climate, particularly the hydrologic cycle. In particular: Could sudden introductions of large amounts of water on the Martian surface lead to a new equilibrated water cycle? Can we tie certain fluvial surface features to transient or sustained water cycles? What are the roles of water vapor and water ice clouds to sudden changes in the water cycle on Mars? How are radiative feedbacks involved with this? What is the ultimate fate of the outflow water? This work uses the NASA Ames MGCM version 2.1 and other schemes that are part of the NASA Ames MGCM suite of tools. Various versions of the MGCM developed at Ames have been used extensively to examine dust and volatile distributions on Mars (e.g., Kahre et al., 2006, 2008). The MGCM 2.1 currently has a well-developed water ice cloud formation scheme (Montmessin et al., 2002, 2004a), which includes calculation of cloud particle concentrations, nucleation, growth, and gravitational sedimentation. For examining the effect of a large water outflow on the climate of Mars, we include water tracers, with an advanced cloud particle scheme Preliminary results suggest that water may have been transported globally for years post-outflow. Post-outflow water cloud formation increases dramatically, with water ice clouds and water vapor potentially transporting water globally. The global mass of water vapor and of water ice clouds increases substantially, with the post-outflow patterns settling into annual cycles, with increasing water entering the atmosphere from the surface over time. Future work will examine the radiative effects of the water vapor and water ice clouds, and the longer-term persistence of a new hydrological or climate regime Detailed comparisons of post-outflow precipitation locations with fluvial features on Mars will be done.
Effect of channel catfish stocking rate on yield and water quality in an intensive, mixed suspended-growth production system
Technology Transfer Automated Retrieval System (TEKTRAN)
This study was conducted to determine the effect of channel catfish (Ictalurus punctatus) stocking rate on yield and water quality in a mixed suspended-growth production system (bio-floc) with zero water exchange. Channel catfish (National Warmwater Aquaculture Center 103 strain; average weight = 13...
The nature of ion and water barrier crossings in a simulated ion channel.
Chiu, S. W.; Novotny, J. A.; Jakobsson, E.
Using a combination of techniques, including molecular dynamics, time-correlation analysis, stochastic dynamics, and fitting of continuum diffusion theory to electrophysiological data, a characterization is made of thermally driven sodium, water, and D2O motion within the gramicidin A channel. Since the channel contents are constrained to move in a single-file fashion, the motion that corresponds to experimentally measurable rates of permeation of the membrane is the motion of the center of mass of the channel contents. We therefore emphasize channel contents center-of-mass motion in our analysis of molecular dynamics computations. The usual free energy calculation techniques would be of questionable validity when applied to such motion. As an alternative to those techniques, we postulate a periodic sinusoidal free energy profile (related to the periodic structure of the helical channel) and deduce the fluid dynamic diffusion coefficient and the height and spacing of the free energy barriers from the form of the mean-square-deviation function, using stochastic computations. The fluid dynamic friction in each case appears similar to that for aqueous solution. However, the diffusive motions are modulated by a spatially periodic free energy profile with a periodicity characteristic of an L-D pair of amino acids in the gramicidin helix, approximately 1.7 A in the model we use. The barrier height depends on which substance is moving in the channel, but in each case is several times thermal energy. For barriers of this width and height, the motion is intermediate between the low-friction (transition-state) and high-friction (Brownian) limits. Thus, neither of these formalisms that have been used commonly to describe membrane permeation gives an accurate picture of the underlying physical process (although the Brownian description seems closer to correct). The non-Markovian Langevin equation must be solved to describe properly the statistics of the process. The "channel state of matter" characteristic of the channel contents appears to have some properties typical of the solid and some typical of the liquid state. The magnitude of the local friction and nature of the ion solvation are similar to the liquid state, but the periodicities of structure, free energy, and dynamics are somewhat solid-like. The alignment of water dipoles in the channel bears some resemblance to the orientational ordering of a nematic liquid crystal, but unlike a nematic liquid crystal, the waters have a degree of translational order as well. Thus, the "channel state" is not adequately described by analogy to either the solid or liquid states or to liquid crystals but must be dealt with as its own characteristic type of condensed matter. PMID:7679301
Pannexin1 Channel Proteins in the Zebrafish Retina Have Shared and Unique Properties
Kurtenbach, Sarah; Prochnow, Nora; Kurtenbach, Stefan; Klooster, Jan; Zoidl, Christiane; Dermietzel, Rolf; Kamermans, Maarten; Zoidl, Georg
In mammals, a single pannexin1 gene (Panx1) is widely expressed in the CNS including the inner and outer retinae, forming large-pore voltage-gated membrane channels, which are involved in calcium and ATP signaling. Previously, we discovered that zebrafish lack Panx1 expression in the inner retina, with drPanx1a exclusively expressed in horizontal cells of the outer retina. Here, we characterize a second drPanx1 protein, drPanx1b, generated by whole-genome duplications during teleost evolution. Homology searches strongly support the presence of pannexin sequences in cartilaginous fish and provide evidence that pannexins evolved whenĀurochordata and chordata evolution split. Further, we confirm Panx1 ohnologs being solely present in teleosts. A hallmark of differential expression of drPanx1a and drPanx1b in various zebrafish brain areas is the non-overlapping protein localization of drPanx1a in the outer and drPanx1b in the inner fish retina. A functional comparison of the evolutionary distant fish and mouse Panx1s revealed both, preserved and unique properties. Preserved functions are the capability to form channels opening at resting potential, which are sensitive to known gap junction and hemichannel blockers, intracellular calcium, extracellular ATP and pH changes. However, drPanx1b is unique due to its highly complex glycosylation pattern and distinct electrophysiological gating kinetics. The existence of two Panx1 proteins in zebrafish displaying distinct tissue distribution, protein modification and electrophysiological properties, suggests that both proteins fulfill different functions in vivo. PMID:24194896
Microfiltration: Effect of retentate protein concentration on limiting flux and serum protein removal with 4-mm-channel ceramic microfiltration membranes.
Hurt, E E; Adams, M C; Barbano, D M
The objective of our study was to determine if the limiting flux and serum protein (SP) removal were different at 8, 9, or 10% true protein (TP) in the microfiltration (MF) retentate recirculation loop using 0.1-Ąm ceramic graded permeability membranes with 4-mm-channel diameters operated at 50░C using a diluted milk protein concentrate with 85% protein on a total solids basis (MPC85) as the MF feed. The limiting flux for the MF of diluted MPC85 was determined at 3 TP concentrations in the recirculation loop (8, 9, and 10%). The experiment was replicated 3 times for a total of 9 runs. On the morning of each run, MPC85 was diluted with reverse osmosis water to an MF feed TP concentration of 5.4%. In all runs, the starting flux was 55kg/m(2) per hour, the flux was increased in steps until the limiting flux was reached. The minimum flux increase was 10kg/m(2) per hour. The limiting flux decreased as TP concentration in the recirculation loop increased. The limiting flux was 154▒0.3, 133▒0.7, and 117▒3.3kg/m(2) per hour at recirculation loop TP concentrations of 8.2▒0.07, 9.2▒0.04, and 10.2▒0.09%, respectively. No effect of recirculation loop TP concentration on the SP removal factor was detected. However, the SP removal factor decreased from 0.80▒0.02 to 0.75▒0.02 as flux was increased from the starting flux of 55kg/m(2) per hour to the limiting flux, with a similar decrease seen at all recirculation loop TP concentrations. PMID:25682139
Free enthalpies of replacing water molecules in protein binding pockets.
Riniker, Sereina; Barandun, Luzi J; Diederich, Franńois; Krõmer, Oliver; Steffen, Andreas; van Gunsteren, Wilfred F
Water molecules in the binding pocket of a protein and their role in ligand binding have increasingly raised interest in recent years. Displacement of such water molecules by ligand atoms can be either favourable or unfavourable for ligand binding depending on the change in free enthalpy. In this study, we investigate the displacement of water molecules by an apolar probe in the binding pocket of two proteins, cyclin-dependent kinase 2 and tRNA-guanine transglycosylase, using the method of enveloping distribution sampling (EDS) to obtain free enthalpy differences. In both cases, a ligand core is placed inside the respective pocket and the remaining water molecules are converted to apolar probes, both individually and in pairs. The free enthalpy difference between a water molecule and a CH(3) group at the same location in the pocket in comparison to their presence in bulk solution calculated from EDS molecular dynamics simulations corresponds to the binding free enthalpy of CH(3) at this location. From the free enthalpy difference and the enthalpy difference, the entropic contribution of the displacement can be obtained too. The overlay of the resulting occupancy volumes of the water molecules with crystal structures of analogous ligands shows qualitative correlation between experimentally measured inhibition constants and the calculated free enthalpy differences. Thus, such an EDS analysis of the water molecules in the binding pocket may give valuable insight for potency optimization in drug design. PMID:23247390
Channel Catfish, Ictalurus punctatus Rafinesque 1818, Tetraspanin Membrane Protein Family: Characterization and Expression Analysis of CD81 cDNA
Technology Transfer Automated Retrieval System (TEKTRAN)
CD81, also known as the target of an antiproliferative antibody 1 (TAPA-1), is a member of tetraspanin integral membrane protein family. This protein plays many important roles in immune functions. In this report, we characterized and analyzed expression of the channel catfish CD81 transcript. T...
Protein tyrosine kinase PYK2 involved in Ca2+-induced regulation of ion channel and MAP kinase functions
S. Lev; H. Moreno; R. Martinez; P. Canoll; E. Peles; J. M. Musacchio; G. D. Plowman; B. Rudy; J. Schlessinger
The protein tyrosine kinase PYK2, which is highly expressed in the central nervous system, is rapidly phosphorylated on tyrosine residues in response to various stimuli that elevate the intracellular calcium concentration, as well as by protein kinase C activation. Activation of PYK2 leads to modulation of ion channel function and activation of the MAP kinase signalling pathway. PYK2 activation may
Crystal structure of an anhydrous form of trehalose: structure of water channels of trehalose polymorphism.
Nagase, H; Ogawa, N; Endo, T; Shiro, M; Ueda, H; Sakurai, M
alpha, alpha-Trehalose (trehalose) is a nonreducing disaccharide of glucose and is accumulated at high concentrations in some anhydrobiotic organisms, which can survive without water for long periods and rapidly resume active metabolism upon hydration. Although it has been proposed that the intriguing mechanism of bioprotection in anhydrobiosis is conferred by a water channel, details of such a channel have yet to be revealed. We determined the crystal structure of a trehalose anhydrate to further understand the relationship between the structure of water channels and the trehalose polymorph. The space group was identical to that of the dihydrate and the lattice constants were also very similar. Among the five intermolecular hydrogen bonds between the trehalose molecules, four were preserved in the anhydrate. If dehydration of the dihydrate is slow and/or gentle enough to preserve the hydrogen bonds, transformation from the dihydrate to the anhydrate may occur. There are two different holes, hole-1 and hole-2, along one crystal axis. Hole-1 is constructed by trehalose molecules with a screw diad at its center, while hole-2 has a smaller diameter and is without a symmetry operator. Because of the screw axis at the center of hole-1, hollows are present at the side of the hole with diameters roughly equal to that of hole-1. Hole-1 and side pockets followed by hollows correspond to the positions of two water molecules of the dihydrate. The side hollows of the water channel are also observed in the water-filled hole of the dihydrate. Consequently, hole-1 is considered to be a one-dimensional water channel with side pockets. We also calculated molecular and crystal energies to examine the rapid water uptake of the anhydrate. It was demonstrated that the intermolecular interactions in the anhydrate were weaker than in the other anhydrous form, and probably also than those in amorphous trehalose. The anhydrate provides water capture for another solid form and gives protection from water uptake. These structural properties of the anhydrate may elucidate bioprotection in anhydrobiosis. PMID:18605683
Monocyte Chemotactic Protein1 Regulates Voltage-Gated K + Channels and Macrophage Transmigration
Howard E. Gendelman; Shengyuan Ding; Nan Gong; Jianuo Liu; Servio H. Ramirez; Yuri Persidsky; R. Lee Mosley; Tong Wang; David J. Volsky; Huangui Xiong
Progressive human immunodeficiency virus (HIV)-1 infection and virus-induced neuroinflammatory responses effectuate monocyte-macrophage\\u000a transmigration across the blood¢brain barrier (BBB). A key factor in mediating these events is monocyte chemotactic protein-1\\u000a (MCP-1). Upregulated glial-derived MCP-1 in HIV-1-infected brain tissues generates a gradient for monocyte recruitment into\\u000a the nervous system. We posit that the inter-relationships between MCP-1, voltage-gated ion channels, cell shape and
Microrheology of protein layers at the air-water interface
NASA Astrophysics Data System (ADS)
Lee, Myung Han; Cardinali, Steven; Reich, Daniel; Stebe, Kathleen; Leheny, Robert
Due to their amphiphilic nature, many proteins in aqueous solution will adsorb at the air-water interface to create a viscoelastic interfacial layer. We present an investigation of the formation and mechanical properties of interfacial protein layers formed by beta-lactoglobulin using microrheological techniques including multiple particle tracking and magnetic nanowire microrheology. We observe the interfacial rheology evolve in time through three stages: (i) an increase in viscosity, (ii) a period of spatial heterogeneity in which the interface contains elastic and viscous regions, and (iii) the development of a uniformly rigid elastic film. We evaluate the dependence of this evolution on the protein-protein interactions, which we tune by varying solution pH. As we will discuss, these studies illustrate the power of microrheological approaches to interfacial rheology.
The Structure and Transport of Water and Hydrated Ions Within Hydrophobic, Nanoscale Channels
Holt, J K; Herberg, J L; Wu, Y; Schwegler, E; Mehta, A
The purpose of this project includes an experimental and modeling investigation into water and hydrated ion structure and transport at nanomaterials interfaces. This is a topic relevant to understanding the function of many biological systems such as aquaporins that efficiently shuttle water and ion channels that permit selective transport of specific ions across cell membranes. Carbon nanotubes (CNT) are model nanoscale, hydrophobic channels that can be functionalized, making them artificial analogs for these biological channels. This project investigates the microscopic properties of water such as water density distributions and dynamics within CNTs using Nuclear Magnetic Resonance (NMR) and the structure of hydrated ions at CNT interfaces via X-ray Absorption Spectroscopy (XAS). Another component of this work is molecular simulation, which can predict experimental measurables such as the proton relaxation times, chemical shifts, and can compute the electronic structure of CNTs. Some of the fundamental questions this work is addressing are: (1) what is the length scale below which nanoscale effects such as molecular ordering become important, (2) is there a relationship between molecular ordering and transport?, and (3) how do ions interact with CNT interfaces? These are questions of interest to the scientific community, but they also impact the future generation of sensors, filters, and other devices that operate on the nanometer length scale. To enable some of the proposed applications of CNTs as ion filtration media and electrolytic supercapacitors, a detailed knowledge of water and ion structure at CNT interfaces is critical.
Use of label-free optical biosensors to detect modulation of potassium channels by G-protein coupled receptors.
Fleming, Matthew R; Shamah, Steven M; Kaczmarek, Leonard K
Ion channels control the electrical properties of neurons and other excitable cell types by selectively allowing ions to flow through the plasma membrane(1). To regulate neuronal excitability, the biophysical properties of ion channels are modified by signaling proteins and molecules, which often bind to the channels themselves to form a heteromeric channel complex(2,3). Traditional assays examining the interaction between channels and regulatory proteins require exogenous labels that can potentially alter the protein's behavior and decrease the physiological relevance of the target, while providing little information on the time course of interactions in living cells. Optical biosensors, such as the X-BODY Biosciences BIND Scanner system, use a novel label-free technology, resonance wavelength grating (RWG) optical biosensors, to detect changes in resonant reflected light near the biosensor. This assay allows the detection of the relative change in mass within the bottom portion of living cells adherent to the biosensor surface resulting from ligand induced changes in cell adhesion and spreading, toxicity, proliferation, and changes in protein-protein interactions near the plasma membrane. RWG optical biosensors have been used to detect changes in mass near the plasma membrane of cells following activation of G protein-coupled receptors (GPCRs), receptor tyrosine kinases, and other cell surface receptors. Ligand-induced changes in ion channel-protein interactions can also be studied using this assay. In this paper, we will describe the experimental procedure used to detect the modulation of Slack-B sodium-activated potassium (KNa) channels by GPCRs. PMID:24562095
Effect of channelization of Rio Puerto Nuevo on ground-water levels in the San Juan metropolitan area, Puerto Rico
Padilla, Ingrid
Channelization and concrete lining of the Rio Puerto Nuevo and its tributaries in the San Juan Metropolitan area has been proposed to control flooding in low lying areas adjacent to the stream. Concern about the effect of these channel modifications on the ground-water system prompted the U.S. Geological Survey in cooperation with the U.S. Army Corps of Engineers to conduct an investigation of surface-water and ground-water interactions in the Rio Puerto Nuevo basin in 1988. A principal objective of this investigation was to determine the potential effect of channelization of the Rio Puerto Nuevo on ground-water levels.
Dynamics of energy distribution in three channel alpha helix protein based on Davydov's ansatz
NASA Astrophysics Data System (ADS)
Ahmad, Faozan; Alatas, Husin
An important aspect of many biological processes at molecular level is the transfer and storage mechanism of bioenergy released in the reaction of the hydrolysis of Adenosinetriphosphate (ATP) by biomacromolecule especially protein. Model of Soliton Davydov is a new break-through that could describe that mechanism. Here we have reformulated quantum mechanical the Davydov theory, using least action principle. Dynamical aspect of the model is analyzed by numerical calculation. We found two dynamical cases: the traveling and pinning soliton that we suggest they are related to the energy transfer and storage mechanism in the protein. Traveling and pinning soliton can be controlled by strength of coupling. In 3- channel approach, we found the breather phenomena in which its frequency is determined by interchannel coupling parameter.
Ring-like pore structures of SecA: Implication for bacterial protein-conducting channels
Wang, Hong-Wei; Chen, Yong; Yang, Hsiuchin; Chen, Xianchuan; Duan, Ming-Xing; Tai, Phang C.; Sui, Sen-Fang
SecA, an essential component of the general protein secretion pathway of bacteria, is present in Escherichia coli as soluble and membrane-integral forms. Here we show by electron microscopy that SecA assumes two characteristic forms in the presence of phospholipid monolayers: dumbbell-shaped elongated structures and ring-like pore structures. The ring-like pore structures with diameters of 8 nm and holes of 2 nm are found only in the presence of anionic phospholipids. These ring-like pore structures with larger 3- to 6-nm holes (without staining) were also observed by atomic force microscopic examination. They do not form in solution or in the presence of uncharged phosphatidylcholine. These ring-like phospholipid-induced pore-structures may form the core of bacterial protein-conducting channels through bacterial membranes. PMID:12642659
Discrete cage form of water hexamer in the hydrophilic channels assembled by heterocyclic azopyrrole
NASA Astrophysics Data System (ADS)
Zhang, Hongwei; Yin, Zhenming
A heterocyclic azopyrrole compound, meso-diethyl-5,5?-bis(thiazolyldiazo)-dipyrromethane (1), has been synthesized and its two crystals have been characterized by X-ray crystallography. The molecules of compound 1 self-assembled into channel structure in the crystal of 1Ę3H2O, whereas interlocked type dimer in the crystal of 1ĘCHCl3. In the 1-D hydrophilic channels of 1Ę3H2O, six water molecules were held together by eight O-HģO hydrogen bonds and formed discrete cage hexameric clusters. The dehydration/rehydration processes of the compound 1 are also studied.
Phosphorylation of the consensus sites of protein kinase A on alpha1D L-type calcium channel.
Ramadan, Omar; Qu, Yongxia; Wadgaonkar, Raj; Baroudi, Ghayath; Karnabi, Eddy; Chahine, Mohamed; Boutjdir, Mohamed
The novel alpha(1D) L-type Ca(2+) channel is expressed in supraventricular tissue and has been implicated in the pacemaker activity of the heart and in atrial fibrillation. We recently demonstrated that PKA activation led to increased alpha(1D) Ca(2+) channel activity in tsA201 cells by phosphorylation of the channel protein. Here we sought to identify the phosphorylated PKA consensus sites on the alpha(1) subunit of the alpha(1D) Ca(2+) channel by generating GST fusion proteins of the intracellular loops, N terminus, proximal and distal C termini of the alpha(1) subunit of alpha(1D) Ca(2+) channel. An in vitro PKA kinase assay was performed for the GST fusion proteins, and their phosphorylation was assessed by Western blotting using either anti-PKA substrate or anti-phosphoserine antibodies. Western blotting showed that the N terminus and C terminus were phosphorylated. Serines 1743 and 1816, two PKA consensus sites, were phosphorylated by PKA and identified by mass spectrometry. Site directed mutagenesis and patch clamp studies revealed that serines 1743 and 1816 were major functional PKA consensus sites. Altogether, biochemical and functional data revealed that serines 1743 and 1816 are major functional PKA consensus sites on the alpha(1) subunit of alpha(1D) Ca(2+) channel. These novel findings provide new insights into the autonomic regulation of the alpha(1D) Ca(2+) channel in the heart. PMID:19074150
Water Behavior in Layered Porous Media with Discrete Flow Channels: Results of a Large-Scale Experiment
R. J. Lenhard; P. Meakin
A meter-scale experimental system (2-m high by 2-m wide by 3-m long) was used to investigate the behavior of water in a model system consisting of two unconsolidated sediment layers separated by a layer containing discrete flow channels. Stainless steel tubes were inserted vertically through a clay matrix to represent the discrete flow-channel layer. The experimental system was well characterized, and results from water infiltration experiments were analyzed. A time series of water arrival at a network of 86 probes located in the unconsolidated sediment layers is presented, as well as water pressure histories at specific locations. Some probes were located at opposite ends of the flow channels to assess water migration through the discrete flow-channel layer. Analyses of the experimental results focused on capillary break phenomena at the interface between the overlying unconsolidated layer and the underlying discrete flow channels. Dissimilar water pressure histories were measured at probes near the upper boundary of the discrete flow-channel layer, suggesting varied and complex water flow behavior. At some locations, a steady or periodic æleakingÆ of water through the discrete flow channels appeared to occur, contrary to capillary break theory. The authors advocate larger-scale experiments to advance our understanding and ability to model fluid flow across a wide range of spatial and temporal scales.
NASA Technical Reports Server (NTRS)
2006-01-01[figure removed for brevity, see original site] Context image for PIA03693 Channel This channel is located south of Iani Chaos. Image information: VIS instrument. Latitude -10.9N, Longitude 345.5E. 17 meter/pixel resolution. Note: this THEMIS visual image has not been radiometrically nor geometrically calibrated for this preliminary release. An empirical correction has been performed to remove instrumental effects. A linear shift has been applied in the cross-track and down-track direction to approximate spacecraft and planetary motion. Fully calibrated and geometrically projected images will be released through the Planetary Data System in accordance with Project policies at a later time. NASA's Jet Propulsion Laboratory manages the 2001 Mars Odyssey mission for NASA's Office of Space Science, Washington, D.C. The Thermal Emission Imaging System (THEMIS) was developed by Arizona State University, Tempe, in collaboration with Raytheon Santa Barbara Remote Sensing. The THEMIS investigation is led by Dr. Philip Christensen at Arizona State University. Lockheed Martin Astronautics, Denver, is the prime contractor for the Odyssey project, and developed and built the orbiter. Mission operations are conducted jointly from Lockheed Martin and from JPL, a division of the California Institute of Technology in Pasadena.
Phylogenetic conservation of protein-lipid motifs in pentameric ligand-gated ion channels.
Barrantes, Francisco J
Using the crosstalk between the nicotinic acetylcholine receptor (nAChR) and its lipid microenvironment as a paradigm, this short overview analyzes the occurrence of structural motifs which appear not only to be conserved within the nAChR family and contemporary eukaryotic members of the pentameric ligand-gated ion channel (pLGIC) superfamily, but also extend to prokaryotic homologues found in bacteria. The evolutionarily conserved design is manifested in: 1) the concentric three-ring architecture of the transmembrane region, 2) the occurrence in this region of distinct lipid consensus motifs in prokaryotic and eukaryotic pLGIC and 3) the key participation of the outer TM4 ring in conveying the influence of the lipid membrane environment to the middle TM1-TM3 ring and this, in turn, to the inner TM2 channel-lining ring, which determines the ion selectivity of the channel. The preservation of these constant structural-functional features throughout such a long phylogenetic span likely points to the successful gain-of-function conferred by their early acquisition. This article is part of a Special Issue entitled: Lipid-protein interactions. PMID:25839355
MBNL and CELF proteins regulate alternative splicing of the skeletal muscle chloride channel CLCN1.
Kino, Yoshihiro; Washizu, Chika; Oma, Yoko; Onishi, Hayato; Nezu, Yuriko; Sasagawa, Noboru; Nukina, Nobuyuki; Ishiura, Shoichi
The expression and function of the skeletal muscle chloride channel CLCN1/ClC-1 is regulated by alternative splicing. Inclusion of the CLCN1 exon 7A is aberrantly elevated in myotonic dystrophy (DM), a genetic disorder caused by the expansion of a CTG or CCTG repeat. Increased exon 7A inclusion leads to a reduction in CLCN1 function, which can be causative of myotonia. Two RNA-binding protein families--muscleblind-like (MBNL) and CUG-BP and ETR-3-like factor (CELF) proteins--are thought to mediate the splicing misregulation in DM. Here, we have identified multiple factors that regulate the alternative splicing of a mouse Clcn1 minigene. The inclusion of exon 7A was repressed by MBNL proteins while promoted by an expanded CUG repeat or CELF4, but not by CUG-BP. Mutation analyses suggested that exon 7A and its flanking region mediate the effect of MBNL1, whereas another distinct region in intron 6 mediates that of CELF4. An exonic splicing enhancer essential for the inclusion of exon 7A was identified at the 5' end of this exon, which might be inhibited by MBNL1. Collectively, these results provide a mechanistic model for the regulation of Clcn1 splicing, and reveal novel regulatory properties of MBNL and CELF proteins. PMID:19720736
IUPHAR-DB: the IUPHAR database of G protein-coupled receptors and ion channels.
Harmar, Anthony J; Hills, Rebecca A; Rosser, Edward M; Jones, Martin; Buneman, O Peter; Dunbar, Donald R; Greenhill, Stuart D; Hale, Valerie A; Sharman, Joanna L; Bonner, Tom I; Catterall, William A; Davenport, Anthony P; Delagrange, Philippe; Dollery, Colin T; Foord, Steven M; Gutman, George A; Laudet, Vincent; Neubig, Richard R; Ohlstein, Eliot H; Olsen, Richard W; Peters, John; Pin, Jean-Philippe; Ruffolo, Robert R; Searls, David B; Wright, Mathew W; Spedding, Michael
The IUPHAR database (IUPHAR-DB) integrates peer-reviewed pharmacological, chemical, genetic, functional and anatomical information on the 354 nonsensory G protein-coupled receptors (GPCRs), 71 ligand-gated ion channel subunits and 141 voltage-gated-like ion channel subunits encoded by the human, rat and mouse genomes. These genes represent the targets of approximately one-third of currently approved drugs and are a major focus of drug discovery and development programs in the pharmaceutical industry. IUPHAR-DB provides a comprehensive description of the genes and their functions, with information on protein structure and interactions, ligands, expression patterns, signaling mechanisms, functional assays and biologically important receptor variants (e.g. single nucleotide polymorphisms and splice variants). In addition, the phenotypes resulting from altered gene expression (e.g. in genetically altered animals or in human genetic disorders) are described. The content of the database is peer reviewed by members of the International Union of Basic and Clinical Pharmacology Committee on Receptor Nomenclature and Drug Classification (NC-IUPHAR); the data are provided through manual curation of the primary literature by a network of over 60 subcommittees of NC-IUPHAR. Links to other bioinformatics resources, such as NCBI, Uniprot, HGNC and the rat and mouse genome databases are provided. IUPHAR-DB is freely available at http://www.iuphar-db.org. PMID:18948278
Grazing Land Management Strongly Controls Water Quality, Sediment and Channel Dynamics in Tallgrass Prairie Headwater Networks
NASA Astrophysics Data System (ADS)
Grudzinski, B. G.; Daniels, M. D.
In the prairie remnants of North America, watershed sediment regimes are heavily influenced by livestock grazing practices. Despite dramatic declines in stream water quality and ecosystem function concomitant with increasing gazing pressures, there have been no studies to quantitatively assess the relationship between various grazing treatments and sediment production in natural grassland ecosystems. In this study, we evaluate suspended sediment transport and channel morphology in the Flint Hills physiographic province using a paired whole-watershed approach, including 2 replicates of high density cattle grazing, 2 replicates of low density cattle grazing, 3 replicates of bison grazing and 3 replicates of no grazing. As expected, results demonstrate that cattle grazing operations increase e-coli, sediment concentrations and increase channel width. However, no significant differences in e-coli, suspended sediment dynamics or channel geomorphology were found between bison grazed and ungrazed watersheds.
Experimental measurement of vapor density in the discharge channel of a pulsed positive streamer discharge in water
NASA Astrophysics Data System (ADS)
Wen, Xiao Qiong; Li, Shu Han; Liu, Jin Yuan; Niu, Zhi Wen
Using the shadowgraph technique, we performed direct measurements of the vapor density in the discharge channel of a pulsed positive streamer discharge in water. With this experimental technique, we quantitatively measured the vapor density in the discharge channel of a pulsed positive streamer discharge in water of conductivity 100 ?S/cm. The results show that the vapor density in the discharge channel ranges from 100 to 660 kg/m3, and presents an increasing tendency along the axial direction of the discharge channel with increasing distance from the tip of the anode.
Studies of the pore-forming domain of a voltage-gated potassium channel protein.
Haris, P I; Ramesh, B; Sansom, M S; Kerr, I D; Srai, K S; Chapman, D
Recent mutagenesis studies have identified a stretch of amino acid residues which form the ion-selective pore of the voltage-gated potassium channel. It has been suggested that this sequence of amino acids forms a beta-barrel structure making up the structure of the ion-selective pore [Hartman, H.A., Kirsch, G.E., Drewe, J.A., Taglialatela, M., Joho, R.H. and Brown, A.M. (1991) Science, 251, 942-944; Yellen, G., Jurman, M.E., Abramson, T. and MacKinnon, R. (1991) Science, 251, 939-942; Yool, A.J. and Schwarz, T.L. (1991) Nature, 349, 700-704]. We have synthesized a polypeptide corresponding to this amino acid sequence (residues 431-449 of the ShA potassium channel from Drosophila). A tetrameric version of this sequence was also synthesized by linking together four of these peptides onto a branching lysine core. Fourier transform infrared (FT-IR) and circular dichroism (CD) spectroscopy have been used to investigate the structure of these peptides after their reconstitution into lyso phosphatidylcholine micelles and lipid bilayers composed of dimyristoyl phosphatidylcholine and dimyristoyl phosphatidylglycerol. The spectroscopic studies show that these peptides are predominantly alpha-helical in these lipid environments. When incorporated into planar lipid bilayers both peptides induce ion channel activity. Molecular modelling studies based upon the propensity of these peptides to form an alpha-helical secondary structure in a hydrophobic environment are described. These results are discussed in the light of recent mutagenesis and binding studies of the Drosophila Shaker potassium ion channel protein. PMID:8170928
Formation of ion-conducting channels by the membrane attack complex proteins of complement.
Shiver, J W; Dankert, J R; Esser, A F
The effects of sequential additions of purified human complement proteins C5b-6, C7, C8, and C9 to assemble the C5b-9 membrane attack complex (MAC) of complement on electrical properties of planar lipid bilayers have been analyzed. The high resistance state of such membranes was impaired after assembly of large numbers of C5b-8 complexes as indicated by the appearance of rapidly fluctuating membrane currents. The C5b-8 induced conductance was voltage dependent and rectifying at higher voltages. Addition of C9 to membranes with very few C5b-8 complexes caused appearance of few discrete single channels of low conductance (5-25 pS) but after some time very large (greater than 0.5 nS) jumps in conductance could be monitored. This high macroscopic conductance state was dominated by 125-pS channels having a lifetime of approximately 1 s. The high conductance state was not stable and declined again after a period of 1-3 h. Incorporation of MAC extracted from complement-lysed erythrocytes into liposomes and subsequent transformation of such complexes into planar bilayers via an intermediate monolayer state resulted in channels with characteristics similar to the ones produced by sequential assembly of C5b-9. Comparison of the high-conductance C5b-9 channel characteristics (lifetime, ion preference, ionic-strength dependence) with those produced by poly(C9) (the circular or tubular aggregation product of C9) as published by Young, J.D.-E., Z.A. Cohn, and E.R. Podack. (1986. Science [Wash. DC]. 233:184-190.) indicates that the two are significantly different. PMID:1720679
Sensitivity of polarization fluctuations to the nature of protein-water interactions: Study of biological water in four different protein-water systems
NASA Astrophysics Data System (ADS)
Ghosh, Rikhia; Banerjee, Saikat; Hazra, Milan; Roy, Susmita; Bagchi, Biman
Since the time of Kirkwood, observed deviations in magnitude of the dielectric constant of aqueous protein solution from that of neat water (ś80) and slower decay of polarization have been subjects of enormous interest, controversy, and debate. Most of the common proteins have large permanent dipole moments (often more than 100 D) that can influence structure and dynamics of even distant water molecules, thereby affecting collective polarization fluctuation of the solution, which in turn can significantly alter solution's dielectric constant. Therefore, distance dependence of polarization fluctuation can provide important insight into the nature of biological water. We explore these aspects by studying aqueous solutions of four different proteins of different characteristics and varying sizes, chicken villin headpiece subdomain (HP-36), immunoglobulin binding domain protein G (GB1), hen-egg white lysozyme (LYS), and Myoglobin (MYO). We simulate fairly large systems consisting of single protein molecule and 20000-30000 water molecules (varied according to the protein size), providing a concentration in the range of ś2-3 mM. We find that the calculated dielectric constant of the system shows a noticeable increment in all the cases compared to that of neat water. Total dipole moment auto time correlation function of water ??MW(0)?MW(t)? is found to be sensitive to the nature of the protein. Surprisingly, dipole moment of the protein and total dipole moment of the water molecules are found to be only weakly coupled. Shellwise decomposition of water molecules around protein reveals higher density of first layer compared to the succeeding ones. We also calculate heuristic effective dielectric constant of successive layers and find that the layer adjacent to protein has much lower value (ś50). However, progressive layers exhibit successive increment of dielectric constant, finally reaching a value close to that of bulk 4-5 layers away. We also calculate shellwise orientational correlation function and tetrahedral order parameter to understand the local dynamics and structural re-arrangement of water. Theoretical analysis providing simple method for calculation of shellwise local dielectric constant and implication of these findings are elaborately discussed in the present work.
The Outer Membrane Protein OmpW Forms an Eight-Stranded beta-Barrel with a Hydrophobic Channel
Hong,H.; Patel, D.; Tamm, L.; van den Berg, B.
Escherichia coli OmpW belongs to a family of small outer membrane (OM) proteins that are widespread in Gram-negative bacteria. Their functions are unknown, but recent data suggest that they may be involved in the protection of bacteria against various forms of environmental stress. In order to gain insight into the function of these proteins we have determined the crystal structure of Escherichia coli OmpW to 2.7 Angstroms resolution. The structure shows that OmpW forms an eight-stranded beta-barrel with a long and narrow hydrophobic channel that contains a bound LDAO detergent molecule. Single channel conductance experiments show that OmpW functions as an ion channel in planar lipid bilayers. The channel activity can be blocked by the addition of LDAO. Taken together, the data suggest that members of the OmpW family could be involved in the transport of small hydrophobic molecules across the bacterial OM.
AG Channel Measurement and Modeling Results for Over-Water and Hilly Terrain Conditions
NASA Technical Reports Server (NTRS)
Matolak, David W.; Sun, Ruoyu
This report describes work completed over the past year on our project, entitled "Unmanned Aircraft Systems (UAS) Research: The AG Channel, Robust Waveforms, and Aeronautical Network Simulations." This project is funded under the NASA project "Unmanned Aircraft Systems (UAS) in the National Airspace System (NAS)." In this report we provide the following: an update on project progress; a description of the over-freshwater and hilly terrain initial results on path loss, delay spread, small-scale fading, and correlations; complete path loss models for the over-water AG channels; analysis for obtaining parameter statistics required for development of accurate wideband AG channel models; and analysis of an atypical AG channel in which the aircraft flies out of the ground site antenna main beam. We have modeled the small-scale fading of these channels with Ricean statistics, and have quantified the behavior of the Ricean K-factor. We also provide some results for correlations of signal components, both intra-band and inter-band. An updated literature review, and a summary that also describes future work, are also included.
ABA signaling in guard cells entails a dynamic protein-protein interaction relay from the PYL-RCAR family receptors to ion channels.
Lee, Sung Chul; Lim, Chae Woo; Lan, Wenzhi; He, Kai; Luan, Sheng
Plant hormone abscisic acid (ABA) serves as an integrator of environmental stresses such as drought to trigger stomatal closure by regulating specific ion channels in guard cells. We previously reported that SLAC1, an outward anion channel required for stomatal closure, was regulated via reversible protein phosphorylation events involving ABA signaling components, including protein phosphatase 2C members and a SnRK2-type kinase (OST1). In this study, we reconstituted the ABA signaling pathway as a protein-protein interaction relay from the PYL/RCAR-type receptors, to the PP2C-SnRK2 phosphatase-kinase pairs, to the ion channel SLAC1. The ABA receptors interacted with and inhibited PP2C phosphatase activity against the SnRK2-type kinase, releasing active SnRK2 kinase to phosphorylate, and activate the SLAC1 channel, leading to reduced guard cell turgor and stomatal closure. Both yeast two-hybrid and bimolecular fluorescence complementation assays were used to verify the interactions among the components in the pathway. These biochemical assays demonstrated activity modifications of phosphatases and kinases by their interaction partners. The SLAC1 channel activity was used as an endpoint readout for the strength of the signaling pathway, depending on the presence of different combinations of signaling components. Further study using transgenic plants overexpressing one of the ABA receptors demonstrated that changing the relative level of interacting partners would change ABA sensitivity. PMID:22935148
Iterative Receiver in Time-Frequency Domain for Shallow Water Acoustic Channel
NASA Astrophysics Data System (ADS)
Zhao, Liang; Ge, Jianhua
Inter-symbol interference (ISI) caused by multi-path propagation, especially in shallow water channel, degrades the performance of underwater acoustic (UWA) communication systems. In this paper, we combine soft minimum mean squared error (MMSE) equalization and the serially concatenated trellis coded modulation (SCTCM) decoding to develop an iterative receiver in time-frequency domain (TFD) for underwater acoustic point to point communications. Based on sound speed profile (SSP) measured in the lake and finite-element ray (FER) tracing method (Bellhop), the shallow water channel is constructed to evaluate the performance of the proposed iterative receiver. The results suggest that the proposed iterative receiver can reduce the calculation complexity of the equalizer and obtain better performance using less receiving elements.
Computations of transmittance and radiance in infrared water vapor sounding channels
NASA Technical Reports Server (NTRS)
Chou, M.-D.
The wing-scaling approximation/k-distribution method, previously developed for computing solar heating rates (Chou and Arking, 1981) was applied to the computation of the transmittance and outgoing radiance in infrared water vapor sounding channels. Functions necessary for the transmittance and radiance computations were computed from molecular line parameters using line-by-line methods. The method was applied to the three HIRS/2 water vapor sounding channels on the TIROS-N satellite, and its accuracy was tested using 11 widely separated atmospheres which ranged from hot-wet tropical atmospheres to cold-dry subarctic atmospheres. Compared to line-by-line calculations, maximum errors were shown to be less than 0.017 in transmittance and 0.4 K in brightness temperature for all cases. The rms errors are less than 0.009 in transmittance and 0.2 K in brightness temperature, the brightness temperature rms error being much smaller than the instrument noise.
Heat shock proteins and p53 play a critical role in K+ channel-mediated tumor cell proliferation and apoptosis.
Han, Xiaobing; Wang, Fang; Yao, Weixing; Xing, Hui; Weng, Danhui; Song, Xiaohong; Chen, Gang; Xi, Ling; Zhu, Tao; Zhou, Jianfeng; Xu, Gang; Wang, Shixuan; Meng, Li; Iadecola, Costantino; Wang, Gang; Ma, Ding
Plasma membrane potassium (K+) channels are required for tumor cell proliferation and apoptosis. However, the signal transduction mechanisms underlying K+ channel-dependent tumor cell proliferation or apoptosis remains elusive. Using HeLa and A2780 cells as study models, we tested the hypothesis that apoptotic proteins are linked with K+ channel-dependent tumor cell cycle and apoptosis. The patch-clamping study using the whole-cell mode revealed two components of voltage-gated outward K+ currents: one is sensitive to either tetraethylammonium (TEA) or tetrandrine (Tet), a maxi-conductance Ca2+-activated K+ (BK) channel blocker, and the other is sensitive to 4-aminopyridine (4-AP), a delayed rectifier K+ channel blocker. MTT and flow cytometry assays showed that TEA, Tet, or iberiotoxin (Ibtx), a selective BK channel blocker, inhibited HeLa and A2780 cell proliferation in a dose-dependent manner with G1 phase arrest. Pretreatment with TEA or Tet also induced apoptosis in HeLa and A2780 cells. However, glibenclamide (Gli), an ATP-sensitive K+ channel blocker, did not influence K+ currents, proliferation or apoptosis. Western blot analyses showed that while pretreatment of TEA and Tet produced an increase in expressions of p53, p21, and Bax, pretreatment of these two agents led to a decrease in expressions of heat shock protein (hsp)90alpha, hsp90beta, and hsp70. Our results indicate that the blockade of BK channels results in tumor cell apoptosis and cycle arrest at G1 phase, and the transduction pathway underlying the anti-proliferative effects is linked to the increased expression of apoptotic protein p53 and the decreased expression of its chaperone proteins hsp. PMID:17624594
Early Fluid and Protein Shifts in Men During Water Immersion
NASA Technical Reports Server (NTRS)
Hinghofer-Szalkay, H.; Harrison, M. H.; Greenleaf, J. E.
High precision blood and plasma densitometry was used to measure transvascular fluid shifts during water immersion to the neck. Six men (28-49 years) undertook 30 min of standing immersion in water at 35.0 +/- 0.2 C; immersion was preceded by 30 min control standing in air at 28 +/- 1 C. Blood was sampled from an antecubital catheter for determination of Blood Density (BD), Plasma Density (PD), Haematocrit (Ht), total Plasma Protein Concentration (PPC), and Plasma Albumin Concentration (PAC). Compared to control, significant decreases (p less than 0.01) in all these measures were observed after 20 min immersion. At 30 min, plasma volume had increased by 11.0 +/- 2.8%; the average density of the fluid shifted from extravascular fluid into the vascular compartment was 1006.3 g/l; albumin moved with the fluid and its albumin concentration was about one-third of the plasma protein concentration during early immersion. These calculations are based on the assumption that the F-cell ratio remained unchanged. No changes in erythrocyte water content during immersion were found. Thus, immersion-induced haemodilution is probably accompanied by protein (mainly albumin) augmentation which accompanies the intra-vascular fluid shift.
Simulations of the effects of water vapor, cloud liquid water, and ice on AMSU moisture channel brightness temperatures
NASA Technical Reports Server (NTRS)
Muller, Bradley M.; Fuelberg, Henry E.; Xiang, Xuwu
Radiative transfer simulations are performed to determine how water vapor and nonprecipitating cloud liquid water and ice particles within typical midlatitude atmospheres affect brightness temperatures T(sub B)'s of moisture sounding channels used in the Advanced Microwave Sounding Unit (AMSU) and AMSU-like instruments. The purpose is to promote a general understanding of passive top-of-atmosphere T(sub B)'s for window frequencies at 23.8, 89.0, and 157.0 GHz, and water vapor frequencies at 176.31, 180.31, and 182.31 GHz by documenting specific examples. This is accomplished through detailed analyses of T(sub B)'s for idealized atmospheres, mostly representing temperate conditions over land. Cloud effects are considered in terms of five basic properties: droplet size distribution, phase, liquid or ice water content, altitude, and thickness. Effects on T(sub B) of changing surface emissivity also are addressed. The brightness temperature contribution functions are presented as an aid to physically interpreting AMSU T(sub B)'s. Both liquid and ice clouds impact the T(sub B)'s in a variety of ways. The T(sub B)'s at 23.8 and 89 GHz are more strongly affected by altostratus liquid clouds than by cirrus clouds for equivalent water paths. In contrast, channels near 157 and 183 GHz are more strongly affected by ice clouds. Higher clouds have a greater impact on 157- and 183-GHz T(sub B)'s than do lower clouds. Clouds depress T(sub B)'s of the higher-frequency channels by suppressing, but not necessarily obscuring, radiance contributions from below. Thus, T(sub B)'s are less closely associated with cloud-top temperatures than are IR radiometric temperatures. Water vapor alone accounts for up to 89% of the total attenuation by a midtropospheric liquid cloud for channels near 183 GHz. The Rayleigh approximation is found to be adequate for typical droplet size distributions; however, Mie scattering effects from liquid droplets become important for droplet size distribution functions with modal radii greater than 20 micrometers near 157 and 183 GHz, and greater than 30-40 micrometers at 89 GHz. This is due mainly to the relatively small concentrations of droplets much larger than the mode radius. Orographic clouds and tropical cumuli have been observed to contain droplet size distributions with mode radii in the 30-40 micrometers range. Thus, as new instruments bridge the gap between microwave and infrared to frequencies even higher than 183 GHz, radiative transfer modelers are cautioned to explicitly address scattering characteristics of such clouds.
Penetration of water vapour into narrow channels during steam sterilization processes
NASA Astrophysics Data System (ADS)
van Doornmalen, J. P. C. M.; Verschueren, M.; Kopinga, K.
In surgery medical devices are used that should be sterile. To obtain surface steam sterilization conditions in hollow medical devices (e.g. endoscopes), sufficient water vapour should be present in the narrow channels in these devices during sterilization. In this paper, a model to calculate the water vapour distribution in narrow channels during steam sterilization processes is presented. The narrow channels in the devices are modelled as tubes with one open and one closed end. The model is restricted to isothermal situations in which no condensation takes place. To validate the model, the time evolution of the water vapour density at the closed end of a test tube is quantified by a pilot experiment based on infrared light absorption measurements. A stainless-steel test tube was used with a length of 54 cm, a radius of 1.5 mm and a wall thickness of 0.5 mm. These dimensions are comparable to the channels in medical instruments. Both the model calculations and the experiments show that for a wide range of sterilization process parameters the vapour density near the closed end of the tube is insufficient for steam sterilization. Despite the simplicity of the model, a fair overall agreement is found between the model predictions and the experimental results. This agreement can be improved significantly by an empirical modification of the boundary conditions at the open end of the tube. Our calculations show that the tube length is the most important parameter. Some possible changes in the process parameters to increase the water vapour concentration at the closed end of the tube are addressed briefly.
Increased transport of antarctic bottom water in the vema channel during the last ice age.
Ledbetter, M T; Johnson, D A
Particle size analyses of surface sediments in the Vema Channel reveal a spatial variation related to the present hydrography. Similar analyses of sediment deposited during the last ice age (18,000 years before the present) indicate a maximum shallowing of the upper limit of Antarctic Bottom Water (AABW) of about 100 meters, coupled with an increase in velocity, which resulted in an increase in AABW transport. PMID:17744187
Trace Elements in Coalbed Methane Produced Water Interacting with Semi-Arid Ephemeral Stream Channels
M. J. Patz; K. J. Reddy; Q. D. Skinner
The objective of this study was to examine the chemistry of trace elements in coalbed methane (CBM) discharge water reacting\\u000a with semi-arid ephemeral stream channels in Powder River Basin, Wyoming. The study area consisted of two ephemeral streams,\\u000a Burger Draw and Sue Draw. These streams are tributaries to the perennial Powder River, Wyoming. Samples were collected bimonthly\\u000a from three CBM
Convergent regulation of skeletal muscle Ca2+ channels by dystrophin, the actin cytoskeleton, and cAMP-dependent protein kinase
NASA Astrophysics Data System (ADS)
Johnson, Barry D.; Scheuer, Todd; Catterall, William A.
The skeletal muscle L-type Ca2+ channel (CaV1.1), which is responsible for initiating muscle contraction, is regulated by phosphorylation by cAMP-dependent protein kinase (PKA) in a voltage-dependent manner that requires direct physical association between the channel and the kinase mediated through A-kinase anchoring proteins (AKAPs). The role of the actin cytoskeleton in channel regulation was investigated in skeletal myocytes cultured from wild-type mice, mdx mice that lack the cytoskeletal linkage protein dystrophin, and a skeletal muscle cell line, 129 CB3. Voltage dependence of channel activation was shifted positively, and potentiation was greatly diminished in mdx myocytes and in 129 CB3 cells treated with the microfilament stabilizer phalloidin. Voltage-dependent potentiation by strong depolarizing prepulses was reduced in mdx myocytes but could be restored by positively shifting the stimulus potentials to compensate for the positive shift in the voltage dependence of gating. Inclusion of PKA in the pipette caused a negative shift in the voltage dependence of activation and restored voltage-dependent potentiation in mdx myocytes. These results show that skeletal muscle Ca2+ channel activity and voltage-dependent potentiation are controlled by PKA and microfilaments in a convergent manner. Regulation of Ca2+ channel activity by hormones and neurotransmitters that use the PKA signal transduction pathway may interact in a critical way with the cytoskeleton and may be impaired by deletion of dystrophin, contributing to abnormal regulation of intracellular calcium concentrations in dystrophic muscle.
A trace component of ginseng that inhibits Ca2+ channels through a pertussis toxin-sensitive G protein.
Nah, S Y; Park, H J; McCleskey, E W
A crude extract from ginseng root inhibits high-threshold, voltage-dependent Ca2+ channels through an unknown receptor linked to a pertussis toxin-sensitive G protein. We now have found the particular compound that seems responsible for the effect: it is a saponin, called ginsenoside Rf (Rf), that is present in only trace amounts within ginseng. At saturating concentrations, Rf rapidly and reversibly inhibits N-type, and other high-threshold, Ca2+ channels in rat sensory neurons to the same degree as a maximal dose of opioids. The effect is dose-dependent (half-maximal inhibition: 40 microM) and it is virtually eliminated by pretreatment of the neurons with pertussis toxin, an inhibitor of G(o) and Gi GTP-binding proteins. Other ginseng saponins--ginsenosides Rb1, Rc, Re, and Rg1--caused relatively little inhibition of Ca2+ channels, and lipophilic components of ginseng root had no effect. Antagonists of a variety of neurotransmitter receptors that inhibit Ca2+ channels fail to alter the effect of Rf, raising the possibility that Rf acts through another G protein-linked receptor. Rf also inhibits Ca2+ channels in the hybrid F-11 cell line, which might, therefore, be useful for molecular characterization of the putative receptor for Rf. Because it is not a peptide and it shares important cellular and molecular targets with opioids, Rf might be useful in itself or as a template for designing additional modulators of neuronal Ca2+ channels. PMID:7568008
Protein kinase C-mediated inhibition of recombinant T-type Cav3.2 channels by neurokinin 1 receptors.
Rangel, Azahel; Sßnchez-Armass, Sergio; Meza, Ulises
The voltage-activated T-type calcium channel (Ca(V)3.2) and the G protein-coupled neurokinin 1 (NK1) receptor are expressed in peripheral tissues and in central neurons, in which they participate in diverse physiological processes, including neurogenic inflammation and nociception. In the present report, we demonstrate that recombinant Ca(V)3.2 channels are reversibly inhibited by NK1 receptors when both proteins are transiently coexpressed in human embryonic kidney 293 cells. We found that the voltage-dependent macroscopic properties of Ca(V)3.2 currents were unaffected during NK1 receptor-mediated inhibition. However, inhibition was attenuated in cells coexpressing either the dominant-negative Galpha(q) Q209L/D277N or the regulator of G protein signaling (RGS) proteins 2 (RGS2) and 3T (RGS3T), which are effective antagonists of Galpha(q/11). By contrast, inhibition was unaffected in cells coexpressing human rod transducin (Galpha(t)), which buffers Gbetagamma. Channel inhibition was blocked by 1-[6-[[17beta-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5-dione (U73122) and bisindolylmaleimide I, selective inhibitors of phospholipase Cbeta and protein kinase C (PKC), respectively. Inhibition was occluded by application of the PKC activator phorbol-12-myristate-13-acetate. Altogether, these data indicate that NK1 receptors inhibit Ca(V)3.2 channels through a voltage-independent signaling pathway that involves Galpha(q/11), phospholipase Cbeta, and PKC. Our results provide novel evidence regarding the mechanisms underlying T-type calcium channel modulation by G protein-coupled receptors. Functional coupling between Ca(V)3.2 channels and NK1 receptors may be relevant in neurogenic inflammation, neuronal rhythmogenesis, nociception, and other physiological processes. PMID:19805509
Comparisons of the hydraulics of water flows in Martian outflow channels with flows of similar scale on earth
NASA Technical Reports Server (NTRS)
Komar, P. D.
The hydraulics of channelized water flows on Mars and the resulting sediment transport rates are calculated, and similar computations are performed for such terrestrial analogs as the Mississippi River and the catastrophic Lake Missoula floods that formed the Channeled Scabland in eastern Washington State. The morphologies of deep-sea channels formed by catastrophic turbidity currents are compared with the Martian channels, many similarities are pointed out, and the hydraulics of the various flows are compared. The results indicate that the velocities, discharges, bottom shear stresses, and sediment-transport capacity of water flows along the Martian channels would be comparable to those of the oceanic turbidity currents and the Lake Missoula floods. It is suggested that the submarine canyons from which turbidity currents originate are the terrestrial counterparts to the chaotic-terrain areas or craters that serve as sources for many of the Martian channels.
Dynamical Coupling of Intrinsically Disordered Proteins and Their Hydration Water: Comparison with Folded Soluble and Membrane Proteins
Gallat, F.-X.; Laganowsky, A.; Wood, K.; Gabel, F.; vanĀEijck, L.; Wuttke, J.; Moulin, M.; Hõrtlein, M.; Eisenberg, D.; Colletier, J.-P.; Zaccai, G.; Weik, M.
Hydration water is vital for various macromolecular biological activities, such as specific ligand recognition, enzyme activity, response to receptor binding, and energy transduction. Without hydration water, proteins would not fold correctly and would lack the conformational flexibility that animates their three-dimensional structures. Motions in globular, soluble proteins are thought to be governed to a certain extent by hydration-water dynamics, yet it is not known whether this relationship holds true for other protein classes in general and whether, in turn, the structural nature of a protein also influences water motions. Here, we provide insight into the coupling between hydration-water dynamics and atomic motions in intrinsically disordered proteins (IDP), a largely unexplored class of proteins that, in contrast to folded proteins, lack a well-defined three-dimensional structure. We investigated the human IDP tau, which is involved in the pathogenic processes accompanying Alzheimer disease. Combining neutron scattering and protein perdeuteration, we found similar atomic mean-square displacements over a large temperature range for the tau protein and its hydration water, indicating intimate coupling between them. This is in contrast to the behavior of folded proteins of similar molecular weight, such as the globular, soluble maltose-binding protein and the membrane protein bacteriorhodopsin, which display moderate to weak coupling, respectively. The extracted mean square displacements also reveal a greater motional flexibility of IDP compared with globular, folded proteins and more restricted water motions on the IDP surface. The results provide evidence that protein and hydration-water motions mutually affect and shape each other, and that there is a gradient of coupling across different protein classes that may play a functional role in macromolecular activity in a cellular context. PMID:22828339
Antibodies to Kv1 potassium channel-complex proteins leucine-rich, glioma inactivated 1 protein and contactin-associated protein-2 in limbic encephalitis, MorvanÆs syndrome and acquired neuromyotonia
Irani, Sarosh R.; Alexander, Sian; Waters, Patrick; Kleopa, Kleopas A.; Pettingill, Philippa; Zuliani, Luigi; Peles, Elior; Buckley, Camilla; Lang, Bethan
Antibodies that immunoprecipitate 125I-?-dendrotoxin-labelled voltage-gated potassium channels extracted from mammalian brain tissue have been identified in patients with neuromyotonia, MorvanÆs syndrome, limbic encephalitis and a few cases of adult-onset epilepsy. These conditions often improve following immunomodulatory therapies. However, the proportions of the different syndromes, the numbers with associated tumours and the relationships with potassium channel subunit antibody specificities have been unclear. We documented the clinical phenotype and tumour associations in 96 potassium channel antibody positive patients (titres >400 pM). Five had thymomas and one had an endometrial adenocarcinoma. To define the antibody specificities, we looked for binding of serum antibodies and their effects on potassium channel currents using human embryonic kidney cells expressing the potassium channel subunits. Surprisingly, only three of the patients had antibodies directed against the potassium channel subunits. By contrast, we found antibodies to three proteins that are complexed with 125I-?-dendrotoxin-labelled potassium channels in brain extracts: (i) contactin-associated protein-2 that is localized at the juxtaparanodes in myelinated axons; (ii) leucine-rich, glioma inactivated 1 protein that is most strongly expressed in the hippocampus; and (iii) Tag-1/contactin-2 that associates with contactin-associated protein-2. Antibodies to Kv1 subunits were found in three sera, to contactin-associated protein-2 in 19 sera, to leucine-rich, glioma inactivated 1 protein in 55 sera and to contactin-2 in five sera, four of which were also positive for the other antibodies. The remaining 18 sera were negative for potassium channel subunits and associated proteins by the methods employed. Of the 19 patients with contactin-associated protein-antibody-2, 10 had neuromyotonia or MorvanÆs syndrome, compared with only 3 of the 55 leucine-rich, glioma inactivated 1 protein-antibody positive patients (P < 0.0001), who predominantly had limbic encephalitis. The responses to immunomodulatory therapies, defined by changes in modified Rankin scores, were good except in the patients with tumours, who all had contactin-associated-2 protein antibodies. This study confirms that the majority of patients with high potassium channel antibodies have limbic encephalitis without tumours. The identification of leucine-rich, glioma inactivated 1 protein and contactin-associated protein-2 as the major targets of potassium channel antibodies, and their associations with different clinical features, begins to explain the diversity of these syndromes; furthermore, detection of contactin-associated protein-2 antibodies should help identify the risk of an underlying tumour and a poor prognosis in future patients. PMID:20663977
Crystal Structure of the Mammalian GIRK2 K+ Channel and Gating Regulation by G-Proteins, PIP2 and Sodium
Whorton, Matthew R.; MacKinnon, Roderick
Summary G-protein-gated K+ channels (Kir3.1¢Kir3.4) control electrical excitability in many different cells. Among their functions relevant to human physiology and disease, they regulate the heart rate and govern a wide range of neuronal activities. Here we present the first crystal structures of a G-protein-gated K+ channel. By comparing the wild-type structure to that of a constitutively active mutant, we identify a global conformational change through which G-proteins could open a G-loop gate in the cytoplasmic domain. The structures of both channels in the absence and presence of PIP2 show that G-proteins open only the G-loop gate in the absence of PIP2, but in the presence of PIP2 the G-loop gate and a second inner helix gate become coupled, so that both gates open. We also identify a strategically located Na+ ion-binding site, which would allow intracellular Na+ to modulate GIRK channel activity. These data provide a mechanistic description of multi-ligand regulation of GIRK channel gating. PMID:21962516
Activation of the epithelial Na+ channel in the collecting duct by vasopressin contributes to water reabsorption.
Bugaj, Vladislav; Pochynyuk, Oleh; Stockand, James D
We used patch-clamp electrophysiology on isolated, split-open murine collecting ducts (CD) to test the hypothesis that regulation of epithelial sodium channel (ENaC) activity is a physiologically important effect of vasopressin. Surprisingly, this has not been tested directly before. We ask whether vasopressin affects ENaC activity distinguishing between acute and chronic effects, as well as, parsing the cellular signaling pathway and molecular mechanism of regulation. In addition, we quantified possible synergistic regulation of ENaC by vasopressin and aldosterone associating this with a requirement for distal nephron Na+ reabsorption during water conservation vs. maintenance of Na+ balance. We find that vasopressin significantly increases ENaC activity within 2-3 min by increasing open probability (P(o)). This activation was dependent on adenylyl cyclase (AC) and PKA. Water restriction (18-24 h) and pretreatment of isolated CD with vasopressin (approximately 30 min) resulted in a similar increase in P(o). In addition, this also increased the number (N) of active ENaC in the apical membrane. Similar to P(o), increases in N were sensitive to inhibitors of AC. Stressing animals with water and salt restriction separately and jointly revealed an important effect of vasopressin: conservation of water and Na+ each independently increased ENaC activity and jointly had a synergistic effect on channel activity. These results demonstrate a quantitatively important action of vasopressin on ENaC suggesting that distal nephron Na+ reabsorption mediated by this channel contributes to maintenance of water reabsorption. In addition, our results support that the combined actions of vasopressin and aldosterone are required to achieve maximally activated ENaC. PMID:19692483
Numerical investigation of interfacial transport resistance due to water droplets in proton exchange membrane fuel cell air channels
Kandlikar, Satish
Nusselt number a b s t r a c t Oxygen transport resistance at the air flow channel and gas diffusion layerNumerical investigation of interfacial transport resistance due to water droplets in proton exchange membrane fuel cell air channels Mustafa Koz a , Satish G. Kandlikar a,b,* a Microsystems
Balanced finite volume WENO and central WENO schemes for the shallow water and the open-channel flow equations
Nelida ?rnjari?-Äic; Senka Vukovic; Luka Sopta
The goal of this work is to extend finite volume WENO and central WENO schemes to the hyperbolic balance laws with geometrical source term and spatially variable flux function. In particular, we apply proposed schemes to the shallow water and the open-channel flow equations where the source term depends on the channel geometry. For obtaining stable numerical schemes that are
Calcium-dependent binding of HCN1 channel protein to hair cell stereociliary tip link protein protocadherin 15 CD3.
Ramakrishnan, Neeliyath A; Drescher, Marian J; Barretto, Roberto L; Beisel, Kirk W; Hatfield, James S; Drescher, Dennis G
The cytoplasmic amino terminus of HCN1, the primary full-length HCN isoform expressed in trout saccular hair cells, was found by yeast two-hybrid protocols to bind the cytoplasmic carboxyl-terminal domain of a protocadherin 15a-like protein. HCN1 was immunolocalized to discrete sites on saccular hair cell stereocilia, consistent with gradated distribution expected for tip link sites of protocadherin 15a. HCN1 message was also detected in cDNA libraries of rat cochlear inner and outer hair cells, and HCN1 protein was immunolocalized to cochlear hair cell stereocilia. As predicted by the trout hair cell model, the amino terminus of rat organ of Corti HCN1 was found by yeast two-hybrid analysis to bind the carboxyl terminus of protocadherin 15 CD3, a tip link protein implicated in mechanosensory transduction. Specific binding between HCN1 and protocadherin 15 CD3 was confirmed with pull-down assays and surface plasmon resonance analysis, both predicting dependence on Ca(2+). In the presence of calcium chelators, binding between HCN1 and protocadherin 15 CD3 was characterized by a K(D) = 2.39 x 10(-7) m. Ca(2+) at 26.5-68.0 microm promoted binding, with K(D) = 5.26 x 10(-8) m (at 61 microm Ca(2+)). Binding by deletion mutants of protocadherin 15 CD3 pointed to amino acids 158-179 (GenBank accession number XP_238200), with homology to the comparable region in trout hair cell protocadherin 15a-like protein, as necessary for binding to HCN1. Amino terminus binding of HCN1 to HCN1, hypothesized to underlie HCN1 channel formation, was also found to be Ca(2+)-dependent, although the binding was skewed toward a lower effective maximum [Ca(2+)] than for the HCN1 interaction with protocadherin 15 CD3. Competition may therefore exist in vivo between the two binding sites for HCN1, with binding of HCN1 to protocadherin 15 CD3 favored between 26.5 and 68 microm Ca(2+). Taken together, the evidence supports a role for HCN1 in mechanosensory transduction of inner ear hair cells. PMID:19008224
Influenza matrix protein 2 alters CFTR expression and function through its ion channel activity
Londino, James D.; Lazrak, Ahmed; Jurkuvenaite, Asta; Collawn, James F.; Noah, James W.
The human cystic fibrosis transmembrane conductance regulator (CFTR) is a cyclic AMP-activated chloride (Cl?) channel in the lung epithelium that helps regulate the thickness and composition of the lung epithelial lining fluid. We investigated whether influenza M2 protein, a pH-activated proton (H+) channel that traffics to the plasma membrane of infected cells, altered CFTR expression and function. M2 decreased CFTR activity in 1) Xenopus oocytes injected with human CFTR, 2) epithelial cells (HEK-293) stably transfected with CFTR, and 3) human bronchial epithelial cells (16HBE14o?) expressing native CFTR. This inhibition was partially reversed by an inhibitor of the ubiquitin-activating enzyme E1. Next we investigated whether the M2 inhibition of CFTR activity was due to an increase of secretory organelle pH by M2. Incubation of Xenopus oocytes expressing CFTR with ammonium chloride or concanamycin A, two agents that alkalinize the secretory pathway, inhibited CFTR activity in a dose-dependent manner. Treatment of M2- and CFTR-expressing oocytes with the M2 ion channel inhibitor amantadine prevented the loss in CFTR expression and activity; in addition, M2 mutants, lacking the ability to transport H+, did not alter CFTR activity in Xenopus oocytes and HEK cells. Expression of an M2 mutant retained in the endoplasmic reticulum also failed to alter CFTR activity. In summary, our data show that M2 decreases CFTR activity by increasing secretory organelle pH, which targets CFTR for destruction by the ubiquitin system. Alteration of CFTR activity has important consequences for fluid regulation and may potentially modify the immune response to viral infection. PMID:23457187
SWELL1, a plasma membrane protein, is an essential component of volume-regulated anion channel
Qiu, Zhaozhu; Dubin, Adrienne E.; Mathur, Jayanti; Tu, Buu; Reddy, Kritika; Miraglia, Loren J.; Reinhardt, J³rgen; Orth, Anthony P.; Patapoutian, Ardem
Summary Maintenance of a constant cell volume in response to extracellular or intracellular osmotic changes is critical for cellular homeostasis. Activation of a ubiquitous volume-regulated anion channel (VRAC) plays a key role in this process; however, its molecular identity in vertebrates remains unknown. Here, we used a cell-based fluorescence assay and performed a genome-wide RNAi screen to find components of VRAC. We identified SWELL1 (LRRC8A), a member of a four-transmembrane protein family with unknown function, as essential for hypotonicity-induced iodide influx. SWELL1 is localized to the plasma membrane, and its knockdown dramatically reduces endogenous VRAC currents and regulatory cell volume decrease in various cell types. Furthermore, point mutations in SWELL1 cause a significant change in VRAC anion selectivity, demonstrating that SWELL1 is an essential VRAC component. These findings enable further molecular characterization of the VRAC channel complex and genetic studies for understanding the function of VRAC in normal physiology and disease. PMID:24725410
Diverse post-translational modifications of the pannexin family of channel-forming proteins
Penuela, Silvia; Lohman, Alexander W; Lai, Wesley; Gyenis, Laszlo; Litchfield, David W; Isakson, Brant E; Laird, Dale W
The pannexin family of channel-forming proteins is composed of 3 distinct but related members called Panx1, Panx2, and Panx3. Pannexins have been implicated in many physiological processes as well as pathological conditions, primarily through their function as ATP release channels. However, it is currently unclear if all pannexins are subject to similar or different post-translational modifications as most studies have focused primarily on Panx1. Using in vitro biochemical assays performed on ectopically expressed pannexins in HEK-293T cells, we confirmed that all 3 pannexins are N-glycosylated to different degrees, but they are not modified by sialylation or O-linked glycosylation in a manner that changes their apparent molecular weight. Using cell-free caspase assays, we also discovered that similar to Panx1, the C-terminus of Panx2 is a substrate for caspase cleavage. Panx3, on the other hand, is not subject to caspase digestion but an in vitro biotin switch assay revealed that it was S-nitrosylated by nitric oxide donors. Taken together, our findings uncover novel and diverse pannexin post-translational modifications suggesting that they may be differentially regulated for distinct or overlapping cellular and physiological functions. PMID:24418849
The Mycobacterium tuberculosis outer membrane channel protein CpnT confers susceptibility to toxic molecules.
Danilchanka, Olga; Pires, David; Anes, Elsa; Niederweis, Michael
Mycobacterium tuberculosis, the causative agent of tuberculosis, is protected from toxic solutes by an effective outer membrane permeability barrier. Recently, we showed that the outer membrane channel protein CpnT is required for efficient nutrient uptake by M. tuberculosis and Mycobacterium bovis BCG. In this study, we found that the cpnT mutant of M. bovis BCG is more resistant than the wild type to a large number of drugs and antibiotics, including rifampin, ethambutol, clarithromycin, tetracycline, and ampicillin, by 8- to 32-fold. Furthermore, the cpnT mutant of M. bovis BCG was 100-fold more resistant to nitric oxide, a major bactericidal agent required to control M. tuberculosis infections in mice. Thus, CpnT constitutes the first outer membrane susceptibility factor in slow-growing mycobacteria. The dual functions of CpnT in uptake of nutrients and mediating susceptibility to toxic molecules are reflected in macrophage infection experiments: while loss of CpnT was detrimental for M. bovis BCG in macrophages that enable bacterial replication, presumably due to inadequate nutrient uptake, it conferred a survival advantage in macrophages that mount a strong bactericidal response. Importantly, the cpnT gene showed a significantly higher density of nonsynonymous mutations in drug-resistant clinical M. tuberculosis strains, indicating that CpnT is under selective pressure in human tuberculosis and/or during chemotherapy. Our results indicate that the CpnT channel constitutes an outer membrane gateway controlling the influx of nutrients and toxic molecules into slow-growing mycobacteria. This study revealed that reducing protein-mediated outer membrane permeability might constitute a new drug resistance mechanism in slow-growing mycobacteria. PMID:25645841
Diabetes induces and calcium channel blockers prevent cardiac expression of proapoptotic thioredoxin-interacting protein.
Chen, Junqin; Cha-Molstad, Hyunjoo; Szabo, Anna; Shalev, Anath
Cardiomyocyte apoptosis is a critical process in the pathogenesis of ischemic and diabetic cardiomyopathy, but the mechanisms are not fully understood. Thioredoxin-interacting protein (TXNIP) has recently been shown to have deleterious effects in the cardiovascular system and we therefore investigated whether it may also play a role in diabetes-associated cardiomyocyte apoptosis. In fact, TXNIP expression was increased in H9C2 cardiomyocytes incubated at high glucose, and cardiac expression of TXNIP and cleaved caspase-3 were also elevated in vivo in streptozotocin- and obesity-induced diabetic mice. Together, these findings not only suggest that TXNIP is involved in diabetic cardiomyopathy but also that it may represent a novel therapeutic target. Surprisingly, testing putative TXNIP modulators revealed that calcium channel blockers reduce cardiomyocyte TXNIP transcription and protein levels in a dose-dependent manner. Oral administration of verapamil for 3 wk also reduced cardiac TXNIP expression in mice even in the face of severe diabetes, and these reduced TXNIP levels were associated with decreased apoptosis. To determine whether lack of TXNIP can mimic the verapamil-induced decrease in apoptosis, we used TXNIP-deficient HcB-19 mice, harboring a natural nonsense mutation in the TXNIP gene. Interestingly, we found significantly reduced cleaved caspase-3 levels in HcB-19 hearts, suggesting that TXNIP plays a critical role in cardiac apoptosis and that the verapamil effects were mediated by TXNIP reduction. Thus our results suggest that TXNIP reduction is a powerful target to enhance cardiomyocyte survival and that agents such as calcium channel blockers may be useful in trying to achieve this goal and prevent diabetic cardiomyopathy. PMID:19258488
Interlayer Water Regulates the Bio-nano Interface of a ?-sheet Protein stacking on Graphene
NASA Astrophysics Data System (ADS)
Lv, Wenping; Xu, Guiju; Zhang, Hongyan; Li, Xin; Liu, Shengju; Niu, Huan; Xu, Dongsheng; Wu, Ren'an
Using molecular dynamics simulations, we investigated an integrated bio-nano interface consisting of a ?-sheet protein stacked onto graphene. We found that the stacking assembly of the model protein on graphene could be controlled by water molecules. The interlayer water filled within interstices of the bio-nano interface could suppress the molecular vibration of surface groups on protein, and could impair the CH...? interaction driving the attraction of the protein and graphene. The intermolecular coupling of interlayer water would be relaxed by the relative motion of protein upon graphene due to the interaction between water and protein surface. This effect reduced the hindrance of the interlayer water against the assembly of protein on graphene, resulting an appropriate adsorption status of protein on graphene with a deep free energy trap. Thereby, the confinement and the relative sliding between protein and graphene, the coupling of protein and water, and the interaction between graphene and water all have involved in the modulation of behaviors of water molecules within the bio-nano interface, governing the hindrance of interlayer water against the protein assembly on hydrophobic graphene. These results provide a deep insight into the fundamental mechanism of protein adsorption onto graphene surface in water.
Hydraulic Processes in Channels of Water-Lain Alluvial Piedmonts in Arid Lands
NASA Astrophysics Data System (ADS)
Vincent, K. R.; Smith, J.
Alluvial piedmonts (including alluvial fans) comprise over 70% of the habitable area of the Southwest and thus flooding on these landforms is important for scientific study and risk assessment. Direct measurement of hydraulic processes operating during geomorphically effective floods is nearly impossible because bankfull flows are short lived and infrequent (recurrence intervals of many decades), and because they inundate complicated dowstream-branching networks of channels and overbank areas. Thus other methods must be employed to understand hydraulic processes. We have made three important observations regarding water-lain alluvial piedmonts in arid lands and the channels on them. First, the channels exhibit a characteristic geometric pattern in which narrow deep reaches repetitively alternate with shallow wide reaches. There is an associated alternation in bed gradient where the steepest beds lead into narrow reaches and the least-steep beds occur at the transition from narrow to wide reaches. Second, flow branches only at the upper half of shallow wide reaches. Third, the piedmonts are hydraulically steep given the available bed material, meaning that the local occurrence of supercritical flow is inevitable. In the present study we hypothesized that the channel expansion/contraction pattern is the result of the spatial alternation of supercritical and subcritical flow, although the reach-averaged Froude number (F) is approximately unity. We tested this by combining 1) field measurements of reconstructed water depth in channels and on nearby overbank areas for an extreme flood that occurred near Tucson, Arizona, 2) topography from post-flood field surveys and pre-flood aerial photography, and 3) a simple hydraulic model for a reach with length of two expansion/contraction wavelengths. In the model, flow was driven by the water surface gradient (imposed by field measurements). A logarithmic velocity structure and quasi-uniform flow were assumed. The input parameter was discharge, and cross sectional area was imposed from the field data. Hydraulic roughness was back calculated from the velocity structure equation for each cross section and was evaluated for reasonableness. The hypothesis that F spatially alternates about critical is clearly demonstrated by this analysis. The hypothesis that the reach-averaged F is unity for bankfull flows in the study reach is consistent with the data. This suggests that the flow hydraulics set the gradient of active alluvial piedmonts. It follows that piedmont gradient, channel dimensions and bed and bank material can be used in conjunction with the reach averaged F=1 criterion to evaluate the nature of flood hazards on hydraulically steep alluvial piedmonts in arid lands without further assumptions about the hydrologic regime.
Vpr Protein of Human Immunodeficiency Virus Type 1 Forms Cation-Selective Channels in Planar Lipid Bilayers
NASA Astrophysics Data System (ADS)
Piller, S. C.; Ewart, G. D.; Premkumar, A.; Cox, G. B.; Gage, P. W.
A small (96-aa) protein, virus protein R (Vpr), of human immunodeficiency virus type 1 contains one hydrophobic segment that could form a membrane-spanning helix. Recombinant Vpr, expressed in Escherichia coli and purified by affinity chromatography, formed ion channels in planar lipid bilayers when it was added to the cis chamber and when the trans chamber was held at a negative potential. The channels were more permeable to Na+ than to Cl- ions and were inhibited when the trans potential was made positive. Similar channel activity was caused by Vpr that had a truncated C terminus, but the potential dependence of channel activity was no longer seen. Antibody raised to a peptide mimicking part of the C terminus of Vpr (AbC) inhibited channel activity when added to the trans chamber but had no effect when added to the cis chamber. Antibody to the N terminus of Vpr (AbN) increased channel activity when added to the cis chamber but had no effect when added to the trans chamber. The effects of potential and antibodies on channel activity are consistent with a model in which the positive C-terminal end of dipolar Vpr is induced to traverse the bilayer membrane when the opposite (trans) side of the membrane is at a negative potential. The C terminus of Vpr would then be available for interaction with AbC in the trans chamber, and the N terminus would be available for interaction with AbN in the cis chamber. The ability of Vpr to form ion channels in vitro suggests that channel formation by Vpr in vivo is possible and may be important in the life cycle of human immunodeficiency virus type 1 and/or may cause changes in cells that contribute to AIDS-related pathologies.
A multi-channel gel electrophoresis and continuous fraction collection apparatus for high throughput protein separation and characterization
Choi, Megan; Nordmeyer, Robert A.; Cornell, Earl; Dong, Ming; Biggin, Mark D.; Jin, Jian
To facilitate a direct interface between protein separation by PAGE and protein identification by mass spectrometry, we developed a multichannel system that continuously collects fractions as protein bands migrate off the bottom of gel electrophoresis columns. The device was constructed using several short linear gel columns, each of a different percent acrylamide, to achieve a separation power similar to that of a long gradient gel. A Counter Free-Flow elution technique then allows continuous and simultaneous fraction collection from multiple channels at low cost. We demonstrate that rapid, high-resolution separation of a complex protein mixture can be achieved on this system using SDS-PAGE. In a 2.5 h electrophoresis run, for example, each sample was separated and eluted into 48-96 fractions over a mass range of 10-150 kDa; sample recovery rates were 50percent or higher; each channel was loaded with up to 0.3 mg of protein in 0.4 mL; and a purified band was eluted in two to three fractions (200 L/fraction). Similar results were obtained when running native gel electrophoresis, but protein aggregation limited the loading capacity to about 50 g per channel and reduced resolution.
TRP channel¢associated factors are a novel protein family that regulates TRPM8 trafficking and activity
Lemonnier, Loic; Shapovalov, George; Gordienko, Dmitri; Poux, Cķline; Bernardini, Michela; Bokhobza, Alexandre; Bidaux, Gabriel; Degerny, Cindy; Verreman, Kathye; Guarmit, Basma; Benahmed, Mohamed; de Launoit, Yvan; Bindels, Rene J.M.; Pla, Alessandra Fiorio; Prevarskaya, Natalia
TRPM8 is a cold sensor that is highly expressed in the prostate as well as in other non-temperature-sensing organs, and is regulated by downstream receptor¢activated signaling pathways. However, little is known about the intracellular proteins necessary for channel function. Here, we identify two previously unknown proteins, which we have named ōTRP channel¢associated factorsö (TCAFs), as new TRPM8 partner proteins, and we demonstrate that they are necessary for channel function. TCAF1 and TCAF2 both bind to the TRPM8 channel and promote its trafficking to the cell surface. However, they exert opposing effects on TRPM8 gating properties. Functional interaction of TCAF1/TRPM8 also leads to a reduction in both the speed and directionality of migration of prostate cancer cells, which is consistent with an observed loss of expression of TCAF1 in metastatic human specimens, whereas TCAF2 promotes migration. The identification of TCAFs introduces a novel mechanism for modulation of TRPM8 channel activity. PMID:25559186
TRP channel-associated factors are a novel protein family that regulates TRPM8 trafficking and activity.
Gkika, Dimitra; Lemonnier, Loic; Shapovalov, George; Gordienko, Dmitri; Poux, Cķline; Bernardini, Michela; Bokhobza, Alexandre; Bidaux, Gabriel; Degerny, Cindy; Verreman, Kathye; Guarmit, Basma; Benahmed, Mohamed; de Launoit, Yvan; Bindels, Rene J M; Fiorio Pla, Alessandra; Prevarskaya, Natalia
TRPM8 is a cold sensor that is highly expressed in the prostate as well as in other non-temperature-sensing organs, and is regulated by downstream receptor-activated signaling pathways. However, little is known about the intracellular proteins necessary for channel function. Here, we identify two previously unknown proteins, which we have named "TRP channel-associated factors" (TCAFs), as new TRPM8 partner proteins, and we demonstrate that they are necessary for channel function. TCAF1 and TCAF2 both bind to the TRPM8 channel and promote its trafficking to the cell surface. However, they exert opposing effects on TRPM8 gating properties. Functional interaction of TCAF1/TRPM8 also leads to a reduction in both the speed and directionality of migration of prostate cancer cells, which is consistent with an observed loss of expression of TCAF1 in metastatic human specimens, whereas TCAF2 promotes migration. The identification of TCAFs introduces a novel mechanism for modulation of TRPM8 channel activity. PMID:25559186
Quantitative analysis of the water occupancy around the selectivity filter of a K+ channel in different gating modes
Weingarth, Markus; van der Cruijsen, Elwin A. W.; Ostmeyer, Jared; Lievestro, Sylke; Roux, BenoŅt; Baldus, Marc
Recovery in K+ channels, i.e., the transition from the inactivated non-conductive selectivity filter conformation towards the conductive conformation, occurs on a timescale of the order of seconds, which is astonishingly long, given that the structural differences among the filter conformations are faint (< 1 ┼). Computational studies and electrophysiological measurements suggested that buried water molecules bound behind the selectivity filter are at the origin of the slowness of recovery in K+ channels. Using a combination of solid-state NMR spectroscopy (ssNMR) and long molecular dynamics simulations, we sketch a high-resolution map of the spatial and temporal distribution of water behind the selectivity filter of a membrane-embedded K+ channel in two different gating modes. Our study demonstrates that buried water molecules with long residence times are spread all along the rear of the inactivated filter, which explains the recovery kinetics. In contrast, the same region of the structure appears to be dewetted when the selectivity filter is in the conductive state. Using proton-detected ssNMR on fully protonated channels, we demonstrate the presence of a pathway that allows for the interchange of buried and bulk water, as required for a functional influence of buried water on recovery and slow inactivation. Furthermore, we provide direct experimental evidence for the presence of additional ordered water molecules that surround the filter and that are modulated by the channelÆs gating mode. PMID:24410583
Protein Solvation in Membranes and at Water-Membrane Interfaces
NASA Technical Reports Server (NTRS)
Pohorille, Andrew; Chipot, Christophe; Wilson, Michael A.
Different salvation properties of water and membranes mediate a host of biologically important processes, such as folding, insertion into a lipid bilayer, associations and functions of membrane proteins. These processes will be discussed in several examples involving synthetic and natural peptides. In particular, a mechanism by which a helical peptide becomes inserted into a model membrane will be described. Further, the molecular mechanism of recognition and association of protein helical segments in membranes will be discussed. These processes are crucial for proper functioning of a cell. A membrane-spanning domain of glycophorin A, which exists as a helical dimer, serves as the model system. For this system, the free energy of dissociation of the helices is being determined for both the wild type and a mutant, in which dimerization is disrupted.
Biodegradable fibre scaffolds incorporating water-soluble drugs and proteins.
Ma, J; Meng, J; Simonet, M; Stingelin, N; Peijs, T; Sukhorukov, G B
A new type of biodegradable drug-loaded fibre scaffold has been successfully produced for the benefit of water-soluble drugs and proteins. Model drug loaded calcium carbonate (CaCO3) microparticles incorporated into poly(lactic acid-co-glycolic acid) (PLGA) fibres were manufactured by co-precipitation of CaCO3 and the drug molecules, followed by electrospinning of a suspension of such drug-loaded microparticles in a PLGA solution. Rhodamine 6G and bovine serum albumin were used as model drugs for our release study, representing small bioactive molecules and protein, respectively. A bead and string structure of fibres was achieved. The drug release was investigated with different drug loadings and in different pH release mediums. Results showed that a slow and sustained drug release was achieved in 40Ādays and the CaCO3 microparticles used as the second barrier restrained the initial burst release. PMID:26155976
Replacement of fish meal in juvenile channel catfish, Ictalurus punctatus, diets using a yeast-derived protein source
Technology Transfer Automated Retrieval System (TEKTRAN)
We examined the effects of a yeast-derived protein source (NuPro) as a replacement for menhaden fish meal on weight gain, specific growth rate (SGR), food conversion ratio (FCR), whole-body composition, and disease resistance in juvenile channel catfish. NuPro replaced 0, 20, 40, 60, 80, and 100% o...
The English Channel: Contamination status of its transitional and coastal waters.
Tappin, A D; Millward, G E
The chemical contamination (organic compounds, metals, radionuclides, microplastics, nutrients) of English Channel waters has been reviewed, focussing on the sources, concentrations and impacts. River loads were only reliable for Pb, whereas atmospheric loads appeared robust for Cd, Pb, Hg, PCB-153 and ?-HCH. Temporal trends in atmospheric inputs were decreasing. Contaminant concentrations in biota were relatively constant or decreasing, but not for Cd, Hg and HBCDD, and deleterious impacts on fish and copepods were reported. However, data on ecotoxicological effects were generally sparse for legacy and emerging contaminants. Intercomparison of activity concentrations of artificial radionuclides in sediments and biota on both Channel coasts was hindered by differences in methodological approaches. Riverine phosphate loads decreased with time, while nitrate loads remained uniform. Increased biomass of algae, attributable to terrestrial inputs of nutrients, has affected benthic production and shellfisheries. A strategic approach to the identification of contaminant impacts on marine biota is recommended. PMID:25649837
Seismic patterns and migration history of submarine fan channels in deep-water area, Niger Delta, West Africa
NASA Astrophysics Data System (ADS)
Zhang, Guotao; Zhang, Shangfeng; Li, Yuan
The channels of deep-water submarine fan under Niger delta slope are characterized by large dimensions special deposition positions and complex formation processes, its geographical location and sedimentary environment also hinder the research and exploration development. According to the strata slicing, RMS amplitude attribute and other techniques, we exhibit the platforms patterns of channels at different period, and based on the analysis of internal architecture and deformation history of channel-leveed systems, migration and evolution process of channel systems could be understood accurately. A great quantity of isolated channels develop in middle Miocene and aggrading streams in late Miocene, which generating because of large scale of turbidity caused by the drop of second order sea-level, which characterized by vertical accretion at smooth channel, while vertical accretion and lateral migration at bend. Evolution of channel systems can be divided into three stages: the initial erosion, erosion and filling alternately, and abandoned stage. With these three stages, the sinuosity of channel change from moderate to high, then decrease. Incision and filling of channels, being during the three development phases, is the driving force of meander-loops migration, which promote three kinds of migration patterns: lateral, down-system and combination migration. The research provides theoretical basis for high-precision prediction and evaluation of deep-water reservoir.
Effects of dietary protein source and protein¢lipid source interaction on channel catfish ( Ictalurus punctatus) egg biochemical composition, egg production and quality, and fry hatching percentage and performance
Todd D. Sink; Rebecca T. Lochmann; Camilo Pohlenz; Alejandro Buentello; Delbert Gatlin III
The relative importance of proteins (amino acids) and protein¢lipid interactions for reproduction of channel catfish can be assessed qualitatively. We conducted a feeding\\/spawning trial to determine the effects of protein source and protein¢lipid interactions of channel catfish broodstock diets on egg biochemical composition and egg and fry production. A general linear mixed model was used to examine statistical significance (?=0.05)
Evidence for Recent Liquid Water on Mars: Channeled Aprons in a Small Crater within Newton Crater
NASA Technical Reports Server (NTRS)
[figure removed for brevity, see original site]Newton Crater is a large basin formed by an asteroid impact that probably occurred more than 3 billion years ago. It is approximately 287 kilometers (178 miles) across. The picture shown here (top) highlights the north wall of a specific, smaller crater located in the southwestern quarter of Newton Crater (above). The crater of interest was also formed by an impact; it is about 7 km (4.4 mi) across, which is about 7 times bigger than the famous Meteor Crater in northern Arizona in North America.The north wall of the small crater has many narrow gullies eroded into it. These are hypothesized to have been formed by flowing water and debris flows. Debris transported with the water created lobed and finger-like deposits at the base of the crater wall where it intersects the floor (bottom center top image). Many of the finger-like deposits have small channels indicating that a liquid--most likely water--flowed in these areas. Hundreds of individual water and debris flow events might have occurred to create the scene shown here. Each outburst of water from higher upon the crater slopes would have constituted a competition between evaporation, freezing, and gravity.The individual deposits at the ends of channels in this MOC image mosaic were used to get a rough estimate of the minimum amount of water that might be involved in each flow event. This is done first by assuming that the deposits are like debris flows on Earth. In a debris flow, no less than about 10% (and no more than 30%) of their volume is water. Second, the volume of an apron deposit is estimated by measuring the area covered in the MOC image and multiplying it by a conservative estimate of thickness, 2 meters (6.5 feet). For a flow containing only 10% water, these estimates conservatively suggest that about 2.5 million liters (660,000 gallons) of water are involved in each event; this is enough to fill about 7 community-sized swimming pools or enough to supply 20 people with their water needs for a year.The MOC high resolution view is located near 41.1oS, 159.8oW and is a mosaic of three different pictures acquired between January and May 2000. The MOC scene is illuminated from the left; north is up. The context picture was acquired in 1977 by the Viking 1 orbiter and is illuminated from the upper right.
Direct interaction between BKCa potassium channel and microtubule-associated protein 1A
Soo Mi Park; Guoxia Liu; Anup Kubal; Matthew Fury; Luxiang Cao; Steven O Marx
The BKCa channel, a potassium channel that is allosterically activated by voltage and calcium, is expressed in both excitable and non-excitable cells. The channel plays an important role in regulating membrane excitability. The channel activity can be modulated by post-translational modifications such as phosphorylation. Recently, hippocampal BKCa channels were shown to be directly modulated by assembly\\/disassembly of the submembranous actin
Generation and phenotype of a transgenic knockout mouse lacking the mercurial-insensitive water channel aquaporin-4.
Ma, T; Yang, B; Gillespie, A; Carlson, E J; Epstein, C J; Verkman, A S
Aquaporin-4 (AQP4) is a mercurial-insensitive, water-selective channel that is expressed in astroglia and basolateral plasma membranes of epithelia in the kidney collecting duct, airways, stomach, and colon. A targeting vector for homologous recombination was constructed using a 7-kb SacI AQP4 genomic fragment in which part of the exon 1 coding sequence was deleted. Analysis of 164 live births from AQP4[+/-] matings showed 41 [+/+], 83 [+/-], and 40 [-/-] genotypes. The [-/-] mice expressed small amounts of a truncated AQP4 transcript and lacked detectable AQP4 protein by immunoblot analysis and immunocytochemistry. Water permeability in an AQP4-enriched brain vesicle fraction in [+/+] mice was high and mercurial insensitive, and was decreased by 14-fold in [-/-] mice. AQP4 deletion did not affect growth or tissue morphology at the light microscopic level. Northern blot analysis showed that tissue-specific expression of AQPs 1, 2, 3, and 5 was not affected by AQP4 deletion. Maximum urine osmolality after a 36-h water deprivation was (in mosM, n = 15) [+/+] 3,342+/-209, [+/-] 3, 225+/-167, and [-/-] 2,616+/-229 (P < 0.025), whereas urine osmolalities before water deprivation did not differ among the genotypes. Rotorod analysis of 35- 38-d-old mice revealed no differences in neuromuscular function (performance time in s, n = 8): [+/+] 297+/-25, [+/-] 322+/-28, [-/-] 288+/-37. These results indicate that AQP4 deletion in CD1 mice has little or no effect on development, survival, growth, and neuromuscular function, but produces a small defect in urinary concentrating ability consistent with its expression in the medullary collecting duct. PMID:9276712
Effects of variations in dietary protein and energy on the growth and survival of channel catfish fry (Ictalurus punctatus)Ā
Niles, Steven Dwight
E PECTS OP VAR IATIONS IN DIETAKE PROTEIN . "0 KNKRGT ON THP. CPQlITE . QiB SClIVIVAL OP CHANNFI CAT" ISIl &ET (ICTAI ORES PCNCTATCS) bl STEVEN "'&NIGET N ILES NiAST EEi Q!i S!C I ETC I EFFECTS OP VARIATIONS TN DIETARY PROTEIN... May, 1982 Major Subject: Wildlife and Fishe"ies Sciences EFFECTS OF VARIATIONS IN DIETARY PROTEIN AND ENERGY ON THE GROWTH AND SURVIVAL OF CHANNEL CATFISH FRY (ICTALURUS PUNCTATUS) A Thesis by STEVEN DWIGHT NILES Approved as to style...
The TRPC Ion Channels: Association with Orai1 and STIM1 Proteins and Participation in Capacitative and Non-capacitative Calcium Entry
Gines M. Salido; Isaac JardĒn; Juan A. Rosado
\\u000a Transient receptor potential (TRP) proteins are involved in a large number of non-selective cation channels that are permeable\\u000a to both monovalent and divalent cations. Two general classes of receptor-mediated Ca2+ entry has been proposed: one of then is conduced by receptor-operated Ca2+ channels (ROC), the second is mediated by channels activated by the emptying of intracellular Ca2+ stores (store-operated channels
Water-surface elevation controls on sediment-transport dynamics in channel-flat environments of intertidal flats
NASA Astrophysics Data System (ADS)
Nowacki, D. J.; Ogston, A. S.
Tidal flats are thought to have a balanced sediment budget between export through channels and import via more diffuse processes over flat boundaries. However, little has been done to understand the mechanisms of sediment transport between channels and flats that span multiple morphological and temporal scales. The muddy flats of southeastern Willapa Bay, Washington, are tidally dominated and receive relatively little direct freshwater influence. We use data from instrumented tripods in representative channel and flat pairs of different orders to a) better understand sediment dynamics in each morphological setting, b) investigate whether sediment fluxes are balanced between channels and flats, and c) determine the importance of channel order on these sediment dynamics. Data from intensive field efforts as well as longer-term deployments help to inform how the hydrodynamic regimes of each environment serve to export or retain sediment and to further characterize the total sediment budget of intertidal flats. Results from two month-long deployments in winter 2009-2010 show channels of all orders in southeastern Willapa Bay were flood dominated. This was driven by longer durations of and sustained higher velocities during flooding tides, and suggests that larger circulation patterns were active within the tidal flat complex. The deployment periods were characterized by a range of meteorological conditions, including rain and several wind events. The wind events were correlated with increased flood dominance of water and sediment transport. Near-bed observations of velocity and suspended-sediment concentration (SSC) give insight to processes active when water levels are shallow over the flat. These processes are important in determining the net flux of water and sediment of the system. High-resolution water-column velocity and backscatter profiles reveal complex sediment-flux dynamics between channel and flat environments. Pulses of velocity and SSC were observed in the channel during flooding and ebbing tides when water levels were near the flat elevation, a phenomenon often observed in tidal flats and salt marshes. Instrumentation deployed near the bed on the flat measured elevated SSC when flat water depth dropped below 10 cm. This ōskimmingö of sediment on the flat contributed to the SSC pulse in the channel during ebbing tides. Water convergence from the flat led to increased channel bottom stresses and resuspension of freshly deposited sediment temporarily stored within the channel. These fine-scale observations allow us to address the mechanisms that govern the total sediment balance of channels and flats within tidal flat systems.
Plumes and Blooms: Modeling the Case II Waters of the Santa Barbara Channel. Chapter 15
NASA Technical Reports Server (NTRS)
Siegel, D. A.; Maritorena, S.; Nelson, N. B.
The goal of the Plumes and Blooms (PnB) project is to develop, validate and apply to imagery state-of-the-art ocean color algorithms for quantifying sediment plumes and phytoplankton blooms for the Case II environment of the Santa Barbara Channel. We conduct monthly to twice-monthly transect observations across the Santa Barbara Channel to develop an algorithm development and product validation data set. The PnB field program started in the summer of 1996. At each of the 7 PnB stations, a complete verification bio-geo-optical data set is collected. Included are redundant measures of apparent optical properties (remote sensing reflectance and diffuse attenuation spectra), as well as in situ profiles of spectral absorption, beam attenuation and backscattering coefficients. Water samples are analyzed for component in vivo absorption spectra, fluorometric chlorophyll, phytoplankton pigment (by the SDSU CHORS laboratory), and inorganic nutrient concentrations. A primary goal is to use the PnB field data set to objectively tune semi-analytical models of ocean color for this site and apply them using available satellite imagery (SeaWiFS and MODIS). In support of this goal, we have also been addressing SeaWiFS ocean color and AVHRR SST imagery. We also are using the PnB data set to address time/space variability of water masses in the Santa Barbara Channel and its relationship to the 1997/1998 El Nino. However, the comparison between PnB field observations and satellite estimates of primary products has been disappointing. We find that field estimates of water-leaving radiance, L(sub wN)(lambda), correspond poorly to satellite estimates for both SeaWiFS and MODIS local area coverage imagery. We believe this is due to poor atmospheric correction due to complex mixtures of aerosol types found in these near-coastal regions. Last, we remain active in outreach activities.
Water Dynamics and Dewetting Transitions in the Small Mechanosensitive Channel MscS
Anishkin, Andriy; Sukharev, Sergei
The dynamics of confined water in capillaries and nanotubes suggests that gating of ion channels may involve not only changes of the pore geometry, but also transitions between water-filled and empty states in certain locations. The recently solved heptameric structure of the small mechanosensitive channel of Escherichia coli, MscS, has revealed a relatively wide (7¢15 ┼) yet highly hydrophobic transmembrane pore. Continuum estimations based on the properties of pore surface suggest low conductance and a thermodynamic possibility of dewetting. To test the predictions we performed molecular dynamics simulations of MscS filled with flexible TIP3P water. Irrespective to the initial conditions, several independent 6-ns simulations converged to the same stable state with the pore water-filled in the wider part, but predominantly empty in the narrow hydrophobic part, displaying intermittent vapor-liquid transitions. The polar gain-of-function substitution L109S in the constriction resulted in a stable hydration of the entire pore. Steered passages of Cl? ions through the narrow part of the pore consistently produced partial ion dehydration and required a force of 200¢400 pN to overcome an estimated barrier of 10¢20 kcal/mole, implying negligibly low conductance. We conclude that the crystal structure of MscS does not represent an open state. We infer that MscS gate, which is similar to that of the nicotinic ACh receptor, involves a vapor-lock mechanism where limited changes of geometry or surface polarity can locally switch the regime between water-filled (conducting) and empty (nonconducting) states. PMID:15111405
Effective three-dimensional superhydrophobic aerogel-coated channel for high efficiency water-droplet transport
NASA Astrophysics Data System (ADS)
Kim, Aeree; Kim, Hyungmo; Lee, Chan; Kim, Joonwon
Three-dimensional superhydrophobic surfaces have been used effectively to optimize droplet transport efficiency in diverse fluidic systems. However, the fabrication methods for superhydrophobic surfaces applicable to fluidic devices usually involve complicated process. Herein, we report a simple but effective method of fabricating a superhydrophobic surface using organically modified silica aerogel. Superhydrophobic aerogel thin film having highly porous micro/nanostructured surface with methyl groups was realized inside a 3D channel by coating it. To demonstrate that the aerogel-coated surface effectively facilitates movement of water droplets, the droplet-based flow characteristics regarding the triple line were conducted.
Liquid Crystalline Properties of Amyloid Protein Fibers in Water
NASA Astrophysics Data System (ADS)
Mezzenga, Raffaele; Jung, Jin-Mi
We have studied the liquid crystalline features of two colloidal systems consisting of food protein amyloid fibrils in water, obtained by heat-denaturation and aggregation of ?-lactoglobulin, a globular dairy protein. The resulting fibrils, have a monodisperse cross section of about 4 nm and two groups of polydisperse contour lengths: (i) fibrils 1-10 ?m long, showing semiflexible polyeletrolyte-like behaviour and (ii) rigid rods 100-200 nm long. In both systems, the fibers are highly charged (+5 e/nm) and stable in water at low ionic strength (0.01 M) and low pH (pH 2). The physical properties of these systems are studied using a polymer physics approach and phase diagrams of these two systems are obtained by changing concentration and pH. Both systems exhibit rich phase behaviours. Interestingly, the experimentally measured isotropic-nematic phase transition was found to occur at concentrations more than one order of magnitude lower than what expected based on Onsager theory. Experimental results are revisited in terms of the Flory theory developed for rigid polymers in solvent of varying conditions.
A Substrate-Fusion Protein Is Trapped inside the Type III Secretion System Channel in Shigella flexneri
Dohlich, Kim; Zumsteg, Anna Brotcke; Goosmann, Christian; Kolbe, Michael
The Type III Secretion System (T3SS) is a macromolecular complex used by Gram-negative bacteria to secrete effector proteins from the cytoplasm across the bacterial envelope in a single step. For many pathogens, the T3SS is an essential virulence factor that enables the bacteria to interact with and manipulate their respective host. A characteristic structural feature of the T3SS is the needle complex (NC). The NC resembles a syringe with a basal body spanning both bacterial membranes and a long needle-like structure that protrudes from the bacterium. Based on the paradigm of a syringe-like mechanism, it is generally assumed that effectors and translocators are unfolded and secreted from the bacterial cytoplasm through the basal body and needle channel. Despite extensive research on T3SS, this hypothesis lacks experimental evidence and the mechanism of secretion is not fully understood. In order to elucidate details of the T3SS secretion mechanism, we generated fusion proteins consisting of a T3SS substrate and a bulky protein containing a knotted motif. Because the knot cannot be unfolded, these fusions are accepted as T3SS substrates but remain inside the NC channel and obstruct the T3SS. To our knowledge, this is the first time substrate fusions have been visualized together with isolated NCs and we demonstrate that substrate proteins are secreted directly through the channel with their N-terminus first. The channel physically encloses the fusion protein and shields it from a protease and chemical modifications. Our results corroborate an elementary understanding of how the T3SS works and provide a powerful tool for in situ-structural investigations in the future. This approach might also be applicable to other protein secretion systems that require unfolding of their substrates prior to secretion. PMID:24453973
Identifying and tracking proteins through the marine water column: insights into the inputs and preservation mechanisms of protein in sediments
Moore, Eli K.; Nunn, Brook L.; Goodlett, David R.; Harvey, H. Rodger
Proteins generated during primary production represent an important fraction of marine organic nitrogen and carbon, and have the potential to provide organism-specific information in the environment. The Bering Sea is a highly productive system dominated by seasonal blooms and was used as a model system for algal proteins to be tracked through the water column and incorporated into detrital sedimentary material. Samples of suspended and sinking particles were collected at multiple depths along with surface sediments on the continental shelf and deeper basin of the Bering Sea. Modified standard proteomic preparations were used in conjunction with high pressure liquid chromatography-tandem mass spectrometry to identify the suite of proteins present and monitor changes in their distribution. In surface waters 207 proteins were identified, decreasing through the water column to 52 proteins identified in post-bloom shelf surface sediments and 24 proteins in deeper (3490 m) basin sediments. The vast majority of identified proteins in all samples were diatom in origin, reflecting their dominant contribution of biomass during the spring bloom. Identified proteins were predominantly from metabolic, binding/structural, and transport-related protein groups. Significant linear correlations were observed between the number of proteins identified and the concentration of total hydrolysable amino acids normalized to carbon and nitrogen. Organelle-bound, transmembrane, photosynthetic, and other proteins involved in light harvesting were preferentially retained during recycling. These findings suggest that organelle and membrane protection represent important mechanisms that enhance the preservation of protein during transport and incorporation into sediments. PMID:22711915
Investigating reaction pathways in rare events simulations of antibiotics diffusion through protein channels.
Hajjar, Eric; Kumar, Amit; Ruggerone, Paolo; Ceccarelli, Matteo
In Gram-negative bacteria, outer-membrane protein channels, such as OmpF of Escherichia coli, constitute the entry point of various classes of antibiotics. While antibacterial research and development is declining, bacterial resistance to antibiotics is rising and there is an emergency call for a new way to develop potent antibacterial agents and to bring them to the market faster and at reduced cost. An emerging strategy is to follow a bottom-up approach based on microscopically founded computational based screening, however such strategy needs better-tuned methods. Here we propose to use molecular dynamics (MD) simulations combined with the metadynamics algorithm, to study antibiotic translocation through OmpF at a molecular scale. This recently designed algorithm overcomes the time scale problem of classical MD by accelerating some reaction coordinates. It is expected that the initial assumption of the reaction coordinates is a key determinant for the efficiency and accuracy of the simulations. Previous studies using different computational schemes for a similar process only used one reaction coordinate, which is the directionality. Here we go further and see how it is possible to include more informative reaction coordinates, accounting explicitly for: (i) the antibiotic flexibility and (ii) interactions with the channel. As model systems, we select two compounds covering the main classes of antibiotics, ampicillin and moxifloxacine. We decipher the molecular mechanism of translocation of each antibiotic and highlight the important parameters that should be taken into account for improving further simulations. This will benefit the screening and design for antibiotics with better permeation properties. PMID:20393764
Water in the Polar and Nonpolar Cavities of the Protein Interleukin-1 Guogang Feng,
Clore, G. Marius
Water in the Polar and Nonpolar Cavities of the Protein Interleukin-1 Hao Yin, Guogang Feng, G ReceiVed: September 13, 2010; ReVised Manuscript ReceiVed: October 19, 2010 Water in the protein by water on the basis of some experiments and simulations and to be empty on the basis of others. Here we
Temperature Dependence of Protein Dynamics Simulated With Three Different Water Models
Glass, Dennis C [ORNL; Krishnan, marimuthu [International Institute of Information Technology, Hyderbad, India; Nutt, David [University of Heidelberg; Smith, Jeremy C [ORNL
Protein and hydration shell dynamics was investigated as function of temperature and water model. Overall dynamics was invariant under the exchange of the water model (TIP3P, TIP4P, TIP5P) for the investigated temperatures (20-300 K). The data provides evidence that changing the water model in protein simulations may be possible without loss of accuracy
Copper accumulation in channel catfish (Ictalurus punctatus) exposed to water borne copper sulfate
Hobbs, M. [Oak Ridge Inst. for Science and Education, TN (United States); Griffin, B. [National Biological Service, Stuttgart, AR (United States); Schlenk, D. [Univ. of Mississippi, Oxford, MS (United States); Kadlubar, F.; Brand, C.D. [National Center for Toxicological Research, Jefferson, AR (United States)
Liver and axial muscle of channel catfish (Ictalurus punctatus) was analyzed for residual copper after exposure to water borne copper sulfate. Copper sulfate was continuously introduced into well water in three fiber glass tanks to achieve 1.7 mg/L, 2.7 mg/L and 3.6 mg/L copper sulfate concentrations in exposure waters. Milli-Q quality water was metered into a fourth tank at the same rate for unexposed fish. Actual levels of copper in exposure waters were determined by daily sampling and analysis by graphite furnace atomic absorption spectrophotometry (GFAA). Tissue samples were taken from six fish from each of the exposed and unexposed tanks at two-week intervals, Samples were collected until tissue analysis indicated an equilibrium had been established between the uptake and elimination in both the muscle and liver tissue. Elimination was followed until a clear rate of deputation could be established. Samples were digested in nitric acid in a micro wave digestor and analyzed by GFAA. Results of tissue analysis will be presented to demonstrate bioaccumulation and the effect of copper concentration, length of copper exposure, and gender on copper uptake, establishment of tissue:environmental copper equilibrium, and rate of copper elimination following exposure.
Effects of water management practices on discharge of nutrients and organic matter from channel catfish ( Ictalurus punctatus) ponds
Craig S Tucker; Susan K Kingsbury; Jonathan W Pole; Charles L Wax
A water quality data set generated by sampling commercial ponds for 2 years was used in a hydrological model of pond overflow volume to describe the effects of water management practices on discharge of nutrients and organic matter from channel catfish Ictalurus punctatus ponds in northwest Mississippi, USA. Average concentrations of total nitrogen, total phosphorus, chemical oxygen demand, and biochemical
Experimental study of Cu-water nanofluid forced convective flow inside a louvered channel
NASA Astrophysics Data System (ADS)
Khoshvaght-Aliabadi, M.; Hormozi, F.; Zamzamian, A.
Heat transfer enhancement plays a very important role for energy saving in plate-fin heat exchangers. In the present study, the influences of simultaneous utilization of a louvered plate-fin channel and copper-base deionized water nanofluid on performance of these exchangers are experimentally explored. The effects of flow rate (2-5 l/min) and nanoparticles weight fraction (0-0.4 %) on heat transfer and pressure drop characteristics are determined. Experimental results indicate that the use of louvered channel instead of the plain one can improve the heat transfer performance. Likewise, addition of small amounts of copper nanoparticles to the base fluid augments the convective heat transfer coefficient remarkably. The maximum rise of 21.7 % in the convective heat transfer coefficient is observed for the 0.4 % wt nanofluid compared to the base fluid. Also, pumping power for the base fluid and nanofluids are calculated based on the measured pressure drop in the louvered channel. The average increase in pumping power is 11.8 % for the nanofluid with 0.4 % wt compared to the base fluid. Applied performance criterion shows a maximum performance index of 1.167 for the nanofluid with 0.1 % wt Finally, two correlations are proposed for Nusselt number and friction factor which fit the experimental data with in ▒10 %.
Conversion of the FhuA transport protein into a diffusion channel through the outer membrane of Escherichia coli.
Killmann, H; Benz, R; Braun, V
The FhuA receptor protein is involved in energy-coupled transport of Fe3+ via ferrichrome through the outer membrane of Escherichia coli. Since no energy source is known in the outer membrane it is assumed that energy is provided through the action of the TonB, ExbB and ExbD proteins, which are anchored to the cytoplasmic membrane. By deleting 34 amino acid residues of a putative cell surface exposed loop, FhuA was converted from a ligand specific transport protein into a TonB independent and nonspecific diffusion channel. The FhuA deletion derivative FhuA delta 322-355 formed stable channels in black lipid membranes, in contrast to wild-type FhuA which did not increase membrane conductance. The single-channel conductance of the FhuA mutant channels was at least three times larger than that of the general diffusion porins of E. coli outer membrane. It is proposed that the basic structure of FhuA in the outer membrane is a channel formed by beta-barrels. Since the loop extending from residue 316 to 356 is part of the active site of FhuA, it probably controls the permeability of the channel. The transport-active conformation of FhuA is mediated by a TonB-induced conformational change in response to the energized cytoplasmic membrane. The ferrichrome transport rate into cells expressing FhuA delta 322-355 increased linearly with increasing substrate concentration (from 0.5 to 20 microM), in contrast to FhuA wild-type cells, which displayed saturation at 5 microM. This implies that in wild-type cells ferrichrome transport through the outer membrane is the rate-limiting step and that TonB, ExbB and ExbD are only required for outer membrane transport. Images PMID:7688295
Pyruvate restores ?-adrenergic sensitivity of L-type Ca2+ channels in failing rat heart: role of protein phosphatase
Zheng, Ming-Qi; Li, Xun; Tang, Kang; Sharma, Neeru M.; Wyatt, Todd A.; Patel, Kaushik P.; Gao, Lie; Bidasee, Keshore R.
Oxidative stress plays a major role in the pathogenesis of heart failure, where the contractile response to ?-adrenergic stimulation is profoundly depressed. This condition involves L-type Ca2+ channels, but the mechanisms underlying their impaired adrenergic regulation are unclear. Thus the present study explored the basis for impaired adrenergic control of Ca2+ channels in a rat infarction model of heart failure. Patch-clamp recordings of L-type Ca2+ current (ICa,L) from ventricular myocytes isolated from infarcted hearts showed a blunted response to intracellular cAMP that was reversed by treatment with exogenous pyruvate. Biochemical studies showed that basal and cAMP-stimulated protein kinase A activities were similar in infarcted and sham-operated hearts, whereas molecular analysis also found that binding of protein kinase A to the ?1C subunit of voltage-gated Ca2+ channel isoform 1.2 was not different between groups. By contrast, protein phosphatase 2A (PP2A) activity and binding to ?1C were significantly less in infarcted hearts. The PP2A inhibitor okadaic acid markedly increased ICa,L in sham-operated myocytes, but this response was significantly less in myocytes from infarcted hearts. However, pyruvate normalized ICa,L stimulation by okadaic acid, and this effect was blocked by inhibitors of thioredoxin reductase, implicating a functional role for the redox-active thioredoxin system. Our data suggest that blunted ?-adrenergic stimulation of ICaL in failing hearts results from hyperphosphorylation of Ca2+ channels secondary to oxidation-induced impairment of PP2A function. We propose that the redox state of Ca2+ channels or PP2A is controlled by the thioredoxin system which plays a key role in Ca2+ channel remodeling of the failing heart. PMID:23504177
Water Permeability of Aquaporin-4 Channel Depends on Bilayer Composition, Thickness, and Elasticity
Tong, Jihong; Briggs, MargaretĀM.; McIntosh, ThomasĀJ.
Aquaporin-4 (AQP4) is the primary water channel in the mammalian brain, particularly abundant in astrocytes, whose plasma membranes normally contain high concentrations of cholesterol. Here we test the hypothesis that the water permeabilities of two naturally occurring isoforms (AQP4-M1 and AQP4-M23) depend on bilayer mechanical/structural properties modulated by cholesterol and phospholipid composition. Osmotic stress measurements were performed with proteoliposomes containing AQP4 and three different lipid mixtures: 1), phosphatidylcholine (PC) and phosphatidylglycerol (PG); 2),ĀPC, PG, with 40Āmol % cholesterol; and 3), sphingomyelin (SM), PG, with 40Āmol % cholesterol. The unit permeabilities of AQP4-M1 were 3.3 ▒ 0.4Āū 10?13 cm3/s (mean ▒ SE), 1.2 ▒ 0.1Āū 10?13 cm3/s, and 0.4 ▒ 0.1Āū 10?13 cm3/s in PC:PG, PC:PG:cholesterol, and SM:PG:cholesterol, respectively. The unit permeabilities of AQP4-M23 were 2.1 ▒ 0.2Āū 10?13 cm3/s, 0.8 ▒ 0.1Āū 10?13 cm3/s, and 0.3 ▒ 0.1Āū 10?13 cm3/s in PC:PG, PC:PG:cholesterol, and SM:PG:cholesterol, respectively. Thus, for each isoform the unit permeabilities strongly depended on bilayer composition and systematically decreased with increasing bilayer compressibility modulus and bilayer thickness. These observations suggest that altering lipid environment provides a means of regulating water channel permeability. Such permeability changes could have physiological consequences, because AQP4 water permeability would be reduced by its sequestration into SM:cholesterol-enriched raft microdomains. Conversely, under ischemic conditions astrocyte membrane cholesterol content decreases, which could increase AQP4 permeability. PMID:23199918
New functions of an old protein: the eukaryotic porin or voltage dependent anion selective channel (VDAC).
De Pinto, Vito; Messina, Angela; Accardi, Rosita; Aiello, Rita; Guarino, Francesca; Tomasello, Marianna Flora; Tommasino, Massimo; Tasco, Gianluca; Casadio, Rita; Benz, Roland; De Giorgi, Francesca; Ichas, Franńois; Baker, Mark; Lawen, Alfons
Mitochondrial porin or VDAC (Voltage Dependent Anion selective Channels) was identified for the first time in 1976, on the basis of the evolutionary similarity between the gram negative and mitochondrial outer membranes. Since this achievement VDAC has been extensively investigated: its functional features have been sharply defined upon reconstitution in artificial membranes and its sequence has been determined in many genomes. Unfortunately the tertiary structure has not yet been solved, mainly because it proved to be very difficult to get suitable crystals. Despite this established knowledge, in the last few years this protein has attracted renewed interest. There are two main reasons for this interest: the discovery, in most eukaryotes, of a family of genes encoding VDAC isoforms and the claims of VDAC involvement in the intrinsic pathway of apoptosis and in particular in the mechanism of cytochrome c release from mitochondria. We can affirm that nowadays the eukaryotic porin (or VDAC) is studied in a more general cellular contest, looking at the interactions and integration with other molecules, since VDAC is in a crucial position in the cell, forming the main interface between the mitochondrial and the cellular metabolisms. In this minireview we will briefly focus our attention onto the following topics: 1) recent advances about the structure of VDAC; 2) the VDAC-related multigene families; 3) the presence, targeting and function of VDAC in various cell membranes. PMID:12833633
The ARH adaptor protein regulates endocytosis of the ROMK potassium secretory channel in mouse kidney
Fang, Liang; Garuti, Rita; Kim, Bo-Young; Wade, James B.; Welling, Paul A.
Renal outer medullary potassium (ROMK) channels are exquisitely regulated to adjust renal potassium excretion and maintain potassium balance. Clathrin-dependent endocytosis plays a critical role, limiting urinary potassium loss in potassium deficiency. In renal disease, aberrant ROMK endocytosis may contribute to potassium retention and hyperkalemia. Previous work has indicated that ROMK endocytosis is stimulated by with-no-lysine (WNK) kinases, but the endocytotic signal and the internalization machinery have not been defined. Here, we found that ROMK bound directly to the clathrin adaptor molecule autosomal recessive hypercholesterolemia (ARH), and this interaction was mediated by what we believe to be a novel variant of the canonical ōNPXYö endocytotic signal, YxNPxFV. ARH recruits ROMK to clathrin-coated pits for constitutive and WNK1-stimuated endocytosis, and ARH knockdown decreased basal rates of ROMK endocytosis, in a heterologous expression system, COS-7 cells. We found that ARH was predominantly expressed in the distal nephron where it coimmunoprecipitated and colocalized with ROMK. In mice, the abundance of kidney ARH protein was modulated by dietary potassium and inversely correlated with changes in ROMK. Furthermore, ARH-knockout mice exhibited an altered ROMK response to potassium intake. These data suggest that ARH marks ROMK for clathrin-dependent endocytosis, in concert with the demands of potassium homeostasis. PMID:19841541
CISD1 codifies a mitochondrial protein upregulated by the CFTR channel.
Taminelli, Guillermo L; Sotomayor, Ver¾nica; Valdivieso, Angel G; Teiber, MarĒa L; MarĒn, MarĒa C; Santa-Coloma, Tomßs A
Cystic fibrosis (CF) is an autosomic recessive disease caused by mutations in the CFTR chloride channel, which indirectly affect the expression of a net of genes. Here we describe a new CFTR-dependent gene, CISD1, encoding for the first member of a family of proteins possessing a CDGSH signature. CISD1 mRNA is down-regulated in cystic fibrosis cells, and restored in the same cells ectopically expressing wt-CFTR (CFDE and CFDE/6RepCFTR; IB3-1 and S9 cells). Inhibition of CFTR chloride transport activity by using glibenclamide (50muM, 24h) or CFTR(inh)-172 (5muM, 24h), resulted in the down-regulation of CISD1 mRNA, and CFTR stimulation with cAMP/isoproterenol/IBMX upregulated its expression. As predicted by PSORT II, a CISD1-GFP chimera was found to be located into mitochondria, suggesting a possible role in the function/regulation of mitochondrial activity, in agreement with earlier observations of a possible mitochondrial failure in cystic fibrosis. PMID:18047834
Water Budget and Modeling of Stream Channel Infiltration of Coalbed Methane Co-Produced Water at a Storage Impoundment Site, Powder River Basin, Wyoming
NASA Astrophysics Data System (ADS)
Payne, A. A.; Saffer, D. M.
Rapid coalbed methane (CBM) development in the Powder River Basin, Wyoming, has resulted in a dramatic increase in the number of producing wells, with as many as 40,000 new wells projected to drilled during the next decade. CBM development involves the co-production of large volumes of coalbed water, which is most commonly discharged to impoundments. Little is known about the potentially significant effects that this co-produced water may have on shallow aquifers and water budgets. Since many of the impoundments in the Powder River Basin are in-channel and supplement surface water flow, it is important to understand what factors influence stream channel infiltration. Modeling of cross-sectional infiltration in a stream channel was undertaken using the U. S. Geological Survey's SUTRA finite-element code, simulating fluid movement from the stream channel, through the unsaturated zone, and into the shallow aquifer. Soil type, saturated hydraulic conductivity, and degree of anisotropy within the soil layer were analyzed to better understand the effects that each have on channel infiltration. At a small study site containing two in-channel infiltration impoundments in the Beaver Creek drainage (a tributary to the Powder River), water budgets have been determined from late July, 2003 to the present (excluding the winter months). Calculated infiltration rates of 0.04 cfs/mile in a 0.8 m wide stream channel compared well with modeled regional soils. Slug tests were utilized to determine aquifer properties in the underlying alluvium/weathered bedrock. A two-layer SUTRA model is presented representing the upper soil layer and underlying alluvium/weathered bedrock and compared to observed changes in groundwater levels in the stream sections affected by introduced CBM water.
Visible-light-induced water-splitting in channels of carbon nanotubes.
Guo, Deng-Zhu; Zhang, Geng-Min; Zhang, Zhao-Xiang; Xue, Zeng-Quan; Gu, Zhen-Nan
The visible-light-induced split of water confined in channels of single-walled carbon nanotubes (SWNTs) was experimentally studied. Arc-discharging synthesized SWNTs were used to adsorb water vapor and then were irradiated in a vacuum by using light from a camera flash. It was found that a great amount of hydrogen-rich gases could be repeatedly produced under several rapid flashes of light, occasionally accompanying evident charge emission phenomena. A quantitative method was developed to estimate the relative amount of gas components on the basis of the data acquired with an ion gauge and a quadrupole mass spectrometer. The results indicated that hydrogen occupied about 80 mol % of the photogenerated gases, with other components such as carbon oxides, helium, methane and trace of ethane, and the total gas yield in one flash (0.1-0.2 J/cm2, 8 ms) reached 400-900 ppm of the mass of the SWNTs. Such a yield could be repeatedly obtained in serial flashings until the adsorbed water was depleted, and then, by sufficiently adsorbing water vapor again, the same phenomena could be reproduced. PMID:16471717
Flow field simulation of gas-water two phase flow in annular channel
NASA Astrophysics Data System (ADS)
Ji, Pengcheng; Dong, Feng
The gas-water two-phase flow is very common in the industrial processes. the deep understanding of the two-phase flow state is to achieve the production equipment design and safe operation. In the measurement of gas-water two-phase flow, the differential pressure sensor is widely used, and some measurement model of multiphase flow have been concluded. The differential pressure is generated when fluid flowing through the throttling components to calculate flow rate. This paper mainly focuses on two points: 1. The change rule of the parameters include velocity, pressure, phase fraction as the change of time, when the phase inlet velocity is given. 2. Analysis the distribution of the parameters above-mentioned at a certain moment under the condition of different water inlet velocity. Three-dimensional computational fluid dynamics (CFD) approach was used to simulate gas-water two-phase flow fluid in the annular channel, which is composed of horizontal pipe and long- waist cone sensor. The simulation results were obtained from FLUENT software.
CRMP2 Protein SUMOylation Modulates NaV1.7 Channel Trafficking*
Dustrude, Erik T.; Wilson, Sarah M.; Ju, Weina; Xiao, Yucheng; Khanna, Rajesh
Voltage-gated sodium channel (NaV) trafficking is incompletely understood. Post-translational modifications of NaVs and/or auxiliary subunits and protein-protein interactions have been posited as NaV-trafficking mechanisms. Here, we tested if modification of the axonal collapsin response mediator protein 2 (CRMP2) by a small ubiquitin-like modifier (SUMO) could affect NaV trafficking; CRMP2 alters the extent of NaV slow inactivation conferred by the anti-epileptic (R)-lacosamide, implying NaV-CRMP2 functional coupling. Expression of a CRMP2 SUMOylation-incompetent mutant (CRMP2-K374A) in neuronal model catecholamine A differentiated (CAD) cells did not alter lacosamide-induced NaV slow inactivation compared with CAD cells expressing wild type CRMP2. Like wild type CRMP2, CRMP2-K374A expressed robustly in CAD cells. Neurite outgrowth, a canonical CRMP2 function, was moderately reduced by the mutation but was still significantly higher than enhanced GFP-transfected cortical neurons. Notably, huwentoxin-IV-sensitive NaV1.7 currents, which predominate in CAD cells, were significantly reduced in CAD cells expressing CRMP2-K374A. Increasing deSUMOylation with sentrin/SUMO-specific protease SENP1 or SENP2 in wild type CRMP2-expressing CAD cells decreased NaV1.7 currents. Consistent with a reduction in current density, biotinylation revealed a significant reduction in surface NaV1.7 levels in CAD cells expressing CRMP2-K374A; surface NaV1.7 expression was also decreased by SENP1 + SENP2 overexpression. Currents in HEK293 cells stably expressing NaV1.7 were reduced by CRMP2-K374A in a manner dependent on the E2-conjugating enzyme Ubc9. No decrement in current density was observed in HEK293 cells co-expressing CRMP2-K374A and NaV1.1 or NaV1.3. Diminution of sodium currents, largely NaV1.7, was recapitulated in sensory neurons expressing CRMP2-K374A. Our study elucidates a novel regulatory mechanism that utilizes CRMP2 SUMOylation to choreograph NaV1.7 trafficking. PMID:23836888
Water management studies in PEM fuel cells, Part II: Ex situ investigation of flow maldistribution, pressure drop and two-phase flow pattern in gas channels
Z. Lu; S. G. Kandlikar; C. Rath; M. Grimm; W. Domigan; A. D. White; M. Hardbarger; J. P. Owejan; T. A. Trabold
Two-phase flow of water and reactant gases in the gas distribution channels of proton exchange membrane fuel cells (PEMFCs) plays a critical role in proper water management. In this work, the two-phase flow in PEMFC cathode parallel channels is studied over a wide range of superficial air velocity (air stoichiometry) and superficial water velocity in a specially designed ex situ
Aquaporins comprise a family of water-transporting membrane proteins. All aquaporins are efficient water transporters, while
de Groot, Bert
. Further insights, particularly with respect to the dynamics of water permeation and the filter mechanism509 Aquaporins comprise a family of water-transporting membrane proteins. All aquaporins are efficient water transporters, while sustaining strict selectivity, even against protons, thereby maintaining
Monitoring water masses properties by Glider in Sardinia Channel during summer 2014
NASA Astrophysics Data System (ADS)
Gana, Slim; Iudicone, Daniele; Ghenim, Leila; Mortier, Laurent; Testor, Pierre; Tintorķ, Joaquin; Olita, Antonio
1. Summary In the framework of the EC funded project, PERSEUS (WP3, Subtask 3.3.1: Repeated glider sections in key channels and sub-basin) and with the support of JERICO TNA (EU-FP7), a deep water glider (up to 1000m) was deployed from the R/V Tethys in the Sardinia Channel and has carried out 3 return trips during the period spanning from the 16th of August 2014 to the 19th of September 2014. The Gilder was equipped with CTD, O2 sensors, Fluorometers (ChlA), back scattering from 470 to 880 nm and was programmed to follow a path close to SARAL satellite track #887. During this experiment, a significant dataset, as never obtained before for this area, has been collected. The innovation stands in the high spatial resolution, in the temporal repetitivity and in the number of parameters sampled simultaneously. The first step of the work will focuses on the analysis of the hydrological properties of the existing water masses in the area. 2. Frame and aim of the experiment The Sardinia Channel is a zonally oriented passage connecting the Algerian and the Tyrrhenian basins, with a sill depth of about 1900 m. In spite of the considerable amount of work achieved and accurate results obtained about the circulation in the Western Mediterranean Sea, during the last 20 years, the Sardinia Channel is still one of the region where the dynamical processes and water exchanges are not clearly identified. Previous studies (Garzoli S. and C. Maillard, 1979, and Ozturgut Erdogan, 1975) pointed out the complexity of the processes in the region and the role of the bottom topography in sustaining them, and provided a first estimation of the involved fluxes. The main knowledge about the water masses crossing this region mostly concerns the AW (Atlantic Water) and the LIW (Levantine Intermediate Water). Along the Algerian coast, the AW is transported mainly by the Algerian current (AC Millot, 1985) from which the anticyclonic Algerian eddies (AEs, Puillat et al., 2002; Taupier-Letage et al., 2003), often involving surface and intermediate waters, are generated by baroclinic instabilities of the AC itself. The AEs generally remain more or less included in the main AC flow. The AEs alongslope-downstream propagation usually ends in the Channel of Sardinia, where AEs dramatically interact with the bathymetry and can remain almost blocked in the Sardinia Channel area for several months before collapsing (Puillat et al., 2002). In order to clarify some of these processes, including the behavior of the Algerian current and associated eddies, our methodology is based on a combined approach using glider observations and sea surface features observed by satellite. By autonomously collecting high-quality observations in three dimensions, gliders allow high-resolution oceanographic monitoring and provide useful contributions for the understanding of mesoscale dynamics and multidisciplinary interactions (e.g., Hodges and Fratantoni, 2009). On top of that, the glider route follows the ground track of the satellite SARAL, equipped with a Ka band altimeter (AltiKa), with the view to implement a methodology of analysis as performed by Bouffard et al. (2010). The main objectives of the project are : Ģ identification of the physical properties of the surface and intermediate water masses between Northern Tunisian Coast and Sardinia and evaluation of the transport of water, salt and heat through the area Ģ study of the variability of the physical properties of surface and intermediate water masses through the use of in-situ and satellite data. Ģ understanding exchanges through sub-basins and the complex interactions through eddies Ģ validation of the operational hydrodynamic numerical model of the western Mediterranean (http://www.seaforecast.cnr.it/en/fl/wmed.php) through the use of in-situ and satellite data. 3. Preliminary results of the experiment The glider carried out 6 legs during the period spanning from the 16th of August 2014 to the 19th of September 2014: Leg#1 (16 to 23 August 2014), Leg#2 (23 to 28 August 2014), Leg#3 (28 Aug. to 03 Sept. 2014)
A Technique For Remote Sensing Of Suspended Sediments And Shallow Coastal Waters Using MODIS Visible and Near-IR Channels
R. Li; Y. Kaufman
ABSTRACT We have developed an algorithm to detect suspended sediments and shallow coastal waters using imaging data acquired with the Moderate Resolution Imaging SpectroRadiometer (MODIS). The MODIS instruments on board the NASA Terra and Aqua Spacecrafts are equipped with one set of narrow channels located in a wide 0.4 - 2.5 micron spectral range. These channels were designed primarily for
Defective interactions of protein partner with ion channels and transporters as alternative mechanisms of membrane channelopathies
Kline, Crystal F.; Mohler, Peter J.
The past twenty years have revealed the existence of numerous ion channel mutations resulting in human pathology. Ion channels provide the basis of diverse cellular functions, ranging from hormone secretion, excitation-contraction coupling, cell signaling, immune response, and trans-epithelial transport. Therefore, the regulation of biophysical properties of channels is vital in human physiology. Only within the last decade has the role of non-ion channel components come to light in regard to ion channel spatial, temporal, and biophysical regulation in physiology. A growing number of auxiliary components have been determined to play elemental roles in excitable cell physiology, with dysfunction resulting in disorders and related manifestations. This review focuses on the broad implications of such dysfunction, focusing on disease-causing mutations that alter interactions between ion channels and auxiliary ion channel components in a diverse set of human excitable cell disease. PMID:23732236
Free lysine ( l-lysine Ę HCl) is utilized for growth less efficiently than protein-bound lysine (soybean meal) in practical diets by young channel catfish ( Ictalurus punctatus)
Dannie D. Zarate; Richard T. Lovell
Two 10-week feeding experiments were conducted with channel catfish fingerlings in aquaria to compare the efficiency of utilization for growth of free versus protein bound lysine in practical diets. In experiment 1, a basal, 26% protein diet, deficient only in lysine, was formulated with a combination of peanut meal and corn gluten meal as the primary protein source. The diet
Potent Neutralization of Influenza A Virus by a Single-Domain Antibody Blocking M2 Ion Channel Protein
Wei, Guowei; Meng, Weixu; Guo, Haijiang; Pan, Weiqi; Liu, Jinsong; Peng, Tao; Chen, Ling; Chen, Chang-You
Influenza A virus poses serious health threat to humans. Neutralizing antibodies against the highly conserved M2 ion channel is thought to offer broad protection against influenza A viruses. Here, we screened synthetic Camel single-domain antibody (VHH) libraries against native M2 ion channel protein. One of the isolated VHHs, M2-7A, specifically bound to M2-expressed cell membrane as well as influenza A virion, inhibited replication of both amantadine-sensitive and resistant influenza A viruses in vitro, and protected mice from a lethal influenza virus challenge. Moreover, M2-7A showed blocking activity for proton influx through M2 ion channel. These pieces of evidence collectively demonstrate for the first time that a neutralizing antibody against M2 with broad specificity is achievable, and M2-7A may have potential for cross protection against a number of variants and subtypes of influenza A viruses. PMID:22164266
Evidence for Recent Liquid Water on Mars: Channels and Aprons in East Gorgonum Crater
NASA Technical Reports Server (NTRS)
[figure removed for brevity, see original site] Warning!This link leads to a very large image that may be too long for some web browsers (in these cases, you must save the link to your desktop and view with other software) [figure removed for brevity, see original site]This suite of Mars Global Surveyor (MGS) Mars Orbiter Camera (MOC) pictures provides a vista of martian gullies on the northern wall of a 12 kilometer-(7.4 mile)-wide meteor impact crater east of the Gorgonum Chaos region on the red planet.The first picture (above left) is a composite of three different high resolution MOC views obtained in 1999 and 2000. The second picture (above right)shows the location of the high resolution views relative to the whole crater as it appeared in the highest resolution image previously acquired of the area, taken by the Viking 1 orbiter in 1978. The release image (top) shows a close-up of one of the channels and debris aprons found in the northwestern quarter of the impact crater.Some of the channels in this crater are deeply-entrenched and cut into lighter-toned deposits. The numerous channels and apron deposits indicate that many tens to hundreds of individual events involving the flow of water and debris have occurred here. The channels and aprons have very crisp, sharp relief and there are no small meteor impact craters on them, suggesting that these features are extremely young relative to the 4.5 billion year history of Mars. It is possible that these landforms are still being created by water seeping from the layered rock in the crater wall today.The crater has no name and it is located near 37.4oS, 168.0oW. The composite view in (above left) includes a picture taken by MOC on September 10, 1999, a picture obtained April 26, 2000, and another on May 22, 2000. The scene from left to right (including the dark gap between photos) covers an area approximately 7.6 kilometers (4.7 miles) wide by 18 km (11.1 mi) long. Sunlight illuminates the scene from the upper left. MOC high resolution images are taken black-and-white (grayscale); the color seen here has been synthesized from the colors of Mars observed by the MOC wide angle cameras and by the Viking Orbiters in the late 1970s.
Interaction of a potential chloride channel blocker with a model transport protein: a spectroscopic and molecular docking investigation.
Ganguly, Aniruddha; Paul, Bijan Kumar; Ghosh, Soumen; Dalapati, Sasanka; Guchhait, Nikhil
The present work demonstrates a detailed characterization of the interaction of a potential chloride channel blocker, 9-methyl anthroate (9-MA), with a model transport protein, Bovine Serum Albumin (BSA). The modulated photophysical properties of the emissive drug molecule within the microheterogeneous bio-environment of the protein have been exploited spectroscopically to monitor the probe-protein binding interaction. Apart from evaluating the binding constant, the probable location of the neutral molecule within the protein cavity (subdomain IB) is explored by an AutoDock-based blind docking simulation. The absence of the Red-Edge Effect has been corroborated by the enhanced lifetime of the probe, being substantially greater than the solvent reorientation time. A dip-and-rise characteristic of the rotational relaxation profile of the drug within the protein has been argued to originate from a significant difference in the lifetime as well as amplitude of the free and protein-bound drug molecule. Unfolding of the protein in the presence of the drug molecule has been probed by the decrease of the ?-helical content, obtained via circular dichroism (CD) spectroscopy, which is also supported by the gradual loss of the esterase activity of the protein in the presence of the drug molecule. PMID:24668158
Protein denaturation and water¢protein interactions as affected by low temperature long time treatment of porcine Longissimus dorsi
Line Christensen; Hanne C. Bertram; Margit D. Aaslyng; Mette Christensen
The relationship between water¢protein interactions and heat-induced protein denaturation in low temperature long time (LTLT) treated pork Longissimus dorsi was investigated by combining low-field NMR T2 relaxometry with DSC measurements and measures of shrinkage of porcine Longissimus dorsi heated to 53░C, 55░C, 57░C and 59░C for either 3 or 20h. Water within the myofibrils, measured by NMR T21 relaxation times,
The major surface protein complex of Treponema denticola depolarizes and induces ion channels in HeLa cell membranes.
Mathers, D A; Leung, W K; Fenno, J C; Hong, Y; McBride, B C
The oral spirochete Treponema denticola is closely associated with periodontal diseases in humans. The 53-kDa major surface protein (Msp) located in the outer membrane of T. denticola serovar a (ATCC 35405) has both pore-forming activity and adhesin activity. We have used standard patch clamp recording methods to study the effects of a partially purified outer membrane complex containing Msp on HeLa cells. The Msp complex was free of the chymotrypsin-like proteinase also found in the outer membrane of T. denticola. Msp bound to several HeLa cell proteins, including a 65-kDa surface protein and a 96-kDa cytoplasmic protein. The Msp complex depolarized and increased the conductance of the HeLa cell membrane in a manner which was not strongly selective for Na+, K+, Ca2+, and Cl- ions. Cell-attached patches of HeLa cell membrane exposed to Msp complex exhibited short-lived channels with a slope conductance of 0.4 nS in physiologically normal saline. These studies show that Msp binds both a putative epithelial cell surface receptor and cytoplasmic proteins and that the Msp complex can form large conductance ion channels in the cytoplasmic membrane of epithelial cells. These properties may contribute to the cytopathic effects of T. denticola on host epithelial cells. PMID:8757811
Molecular characterization, phylogenetic analysis and expression patterns of five protein arginine methyltransferase genes of channel catfish, Ictalurus punctatus (Rafinesque).
Yeh, Hung-Yueh; Klesius, Phillip H
Protein arginine methylation, catalyzed by protein arginine methyltransferases (PRMT), has recently emerged as an important modification in the regulation of gene expression. In this communication, we identified and characterized the channel catfish orthologs to human PRMT 1, 3, 4 and 5, and PRMT4 like. Each PRMT nucleic acid sequence has an open reading frame (ORF) and 3'-untranslated regions. Each ORF appears to encode 361, 587 and 458 amino acid residues for PRMT1, PRMT4 and variant, respectively. The partial ORF of PRMT3 and PRMT5 encode 292 and 563 amino acids, respectively. By comparison with the human counterparts, each channel catfish PRMT also has conserved domains. For expression profile, the channel catfish PRMT1 transcript was detected by RT-PCR in spleens, anterior kidneys, livers, intestines, skin and gills of fish examined. Except in liver, the PRMT3 transcript was detected in all catfish tissues examined. However, the PRMT4 cDNA was detected in livers from all three catfish and gills from two fish, but not other tissues. This information will enable us to further elucidate PRMT functions in channel catfish. PMID:22286871
The mechanosensory protein MEC-6 is a subunit of the C. elegans touch-cell degenerin channel
Dattananda S. Chelur; Glen G. Ernstrom; Miriam B. Goodman; C. Andrea Yao; Lei Chen; Robert O'Hagan; Martin Chalfie
Mechanosensory transduction in touch receptor neurons is believed to be mediated by DEG\\/ENaC (degenerin\\/epithelial Na+ channel) proteins in nematodes and mammals. In the nematode Caenorhabditis elegans, gain-of-function mutations in the degenerin genes mec-4 and mec-10 (denoted mec-4(d) and mec-10(d), respectively) cause degeneration of the touch cells. This phenotype is completely suppressed by mutation in a third gene, mec-6 (refs 3,
Incorporation of the gene for a cell-cell channel protein into transformed cells leads to normalization of growth
Parmender P. Mehta; Agnes Hotz-Wagenblatt; Birgit Rose; David Shalloway; Warner R. Loewenstein
Summary Incorporation of the gene for connexin 43, a cell-cell channel protein of gap junction, into the genome of communication-deficient transformed mouse 10T1\\/2 cells restored junctional communication and inhibited growth. Growth was slowed, saturation density reduced and focus formation suppressed, and these effects were contingent on overexpression of the exogenous gene and the consequent enhancement of communication. In coculture with
G Protein-Coupled Inwardly Rectifying K + Channels (GIRKs) Mediate Postsynaptic but Not Presynaptic Transmitter Actions in Hippocampal Neurons
Christian L³scher; Lily Y Jan; Markus Stoffel; Robert C Malenka; Roger A Nicoll
To study the role of G protein-coupled, inwardly rectifying K+ (GIRK) channels in mediating neurotransmitter actions in hippocampal neurons, we have examined slices from transgenic mice lacking the GIRK2 gene. The outward currents evoked by agonists for GABAB receptors, 5HT1A receptors, and adenosine A1 receptors were essentially absent in mutant mice, while the inward current evoked by muscarinic receptor activation
Mechanism of Interaction between the General Anesthetic Halothane and a Model Ion Channel Protein, II: Fluorescence and Vibrational Spectroscopy Using a Cyanophenylalanine Probe
Liu, J.; Strzalka, J; Tronin, A; Johansson, J; Blasie, J
We demonstrate that cyano-phenylalanine (PheCN) can be utilized to probe the binding of the inhalational anesthetic halothane to an anesthetic-binding, model ion channel protein hbAP-PheCN. The Trp to PheCN mutation alters neither the a-helical conformation nor the 4-helix bundle structure. The halothane binding properties of this PheCN mutant hbAP-PheCN, based on fluorescence quenching, are consistent with those of the prototype, hbAP1. The dependence of fluorescence lifetime as a function of halothane concentration implies that the diffusion of halothane in the nonpolar core of the protein bundle is one-dimensional. As a consequence, at low halothane concentrations, the quenching of the fluorescence is dynamic, whereas at high concentrations the quenching becomes static. The 4-helix bundle structure present in aqueous detergent solution and at the air-water interface, is preserved in multilayer films of hbAP-PheCN, enabling vibrational spectroscopy of both the protein and its nitrile label (-CN). The nitrile groups' stretching vibration band shifts to higher frequency in the presence of halothane, and this blue-shift is largely reversible. Due to the complexity of this amphiphilic 4-helix bundle model membrane protein, where four PheCN probes are present adjacent to the designed cavity forming the binding site within each bundle, all contributing to the infrared absorption, molecular dynamics (MD) simulation is required to interpret the infrared results. The MD simulations indicate that the blue-shift of -CN stretching vibration induced by halothane arises from an indirect effect, namely an induced change in the electrostatic protein environment averaged over the four probe oscillators, rather than a direct interaction with the oscillators. hbAP-PheCN therefore provides a successful template for extending these investigations of the interactions of halothane with the model membrane protein via vibrational spectroscopy, using cyano-alanine residues to form the anesthetic binding cavity.
The origin of proteins: Heteropolypeptides from hydrogen cyanide and water.
Matthews, C N
Evidence from laboratory and extraterrestrial chemistry is presented consistent with the hypothesis that the original heteropolypeptides on Earth were synthesized spontaneously from hydrogen cyanide and water without the intervening formation of chi-amino acids, a key step being the direct polymerization of atmospheric hydrogen cyanide to polyaminomalononitrile (IV) via dimeric HCN. Molecular orbital calculations (INDO) show that the most probable structure for (HCN)2 is azacyclopropenylidenimine. Successive reactions of hydrogen cyanide with the reactive nitrile side chains of IV then yield heteropolyamidines which are converted by water to heteropolypeptides. To study this postulated modification of a homopolymer to a heteropolymer, poly-chi-cyanoglycine (IX) was prepared from the N-carboxyanhydride of chi-cyanoglycine. Hydrolysis of IX, a polyamide analog of the polyamidine IV, yielded glycine. However, when IX was hydrolysed after being treated with hydrogen cyanide, other chi-amino acids were also obtained including alanine, serine, aspartic acid and glutamic acid, suggesting that the nitrile groups of IX (and therfore of IV) are indeed readily attacked by hydrogen cyanide as predicted. Further theoretical and experimental studies support the view that hydrogen cyanide polymerization along these lines is a universal process that accounts not only for the past formation of primitive proteins on Earth, but also for the yellow-brown-orange colors of Jupiter today and for the presence of water-soluble compounds hydrolyzable to chi-amino acids in materials obtained from environments as diverse as the moon, carbonaceous chondrites and the reaction chambers used to simulate organic synthesis in planetary atmospheres. PMID:168535
Fragile X mental retardation protein controls synaptic vesicle exocytosis by modulating N-type calcium channel density
NASA Astrophysics Data System (ADS)
Ferron, Laurent; Nieto-Rostro, Manuela; Cassidy, John S.; Dolphin, Annette C.
Fragile X syndrome (FXS), the most common heritable form of mental retardation, is characterized by synaptic dysfunction. Synaptic transmission depends critically on presynaptic calcium entry via voltage-gated calcium (CaV) channels. Here we show that the functional expression of neuronal N-type CaV channels (CaV2.2) is regulated by fragile X mental retardation protein (FMRP). We find that FMRP knockdown in dorsal root ganglion neurons increases CaV channel density in somata and in presynaptic terminals. We then show that FMRP controls CaV2.2 surface expression by targeting the channels to the proteasome for degradation. The interaction between FMRP and CaV2.2 occurs between the carboxy-terminal domain of FMRP and domains of CaV2.2 known to interact with the neurotransmitter release machinery. Finally, we show that FMRP controls synaptic exocytosis via CaV2.2 channels. Our data indicate that FMRP is a potent regulator of presynaptic activity, and its loss is likely to contribute to synaptic dysfunction in FXS.
A conceptual model for river water and sediment dispersal in the Santa Barbara Channel, California
Warrick, J.A.; Mertes, L.A.K.; Washburn, L.; Siegel, D.A.
The ephemeral Santa Clara River delivers large amounts of freshwater and sediment to the eastern Santa Barbara Channel during brief, episodic discharge events. This discharge into the channel was characterized here with shipboard measurements during floods of 1997 and 1998. Within approximately 1-km of the river mouth, the river discharge quickly stratifies into a freshened, turbid surface plume and a bottom nephloid layer. Observations immediately off the Santa Clara River mouth on a peak day of river discharge revealed that sediment rapidly settled from the freshened surface waters, as suspended sediment in the freshened surface plume contained only ???6% of the sediment mass expected if the sediment mixed conservatively. On the two subsequent days the reduction of sediment mass in the surface plume continued at ???50% per day. These observations suggest that river sediment undergoes rapid initial settling within ???1-km of the river mouth, followed by somewhat slower rates of settling. Although we did not measure sedimentation or bottom boundary layer processes, our mass balance results suggest that almost all of the river sediment either escapes along or deposits upon the inner shelf seabed.
A device to achieve low Reynolds numbers in an open surface water channel
NASA Astrophysics Data System (ADS)
Radi, Alexander; Lo Jacono, David; Sheridan, John
When investigating flow structures, and especially flow transitions, research projects often seek to increase insight using complementary numerical and physical experiments. Obtaining exact Reynolds number correspondence can frequently be difficult in experiments, particularly when relatively low values are required. Often, available test facilities were designed and optimised for a specific velocity range, meaning they have restrictions on the minimum flow velocity. This study describes a device to reduce the flow velocity locally in an open surface water channel. The underlying idea is to divert a controlled fraction of the incoming flow from the working section by increasing the pressure there, resulting in reduced velocity. This idea is realised using a `sub-channel' that can be inserted into the main test chamber, with a variable porosity perforated screen at its downstream end. This study assesses and optimises the flow quality inside this structure, such as usable test section length, uniformity of the velocity profiles and turbulence intensity. The results demonstrate that the device creates high quality low Reynolds number flows, which is exemplified with the canonical circular cylinder in cross-flow.
Steady-State Modulation of Voltage-Gated K+ Channels in Rat Arterial Smooth Muscle by Cyclic AMP-Dependent Protein Kinase and Protein Phosphatase 2B
Brignell, Jennifer L.; Perry, Matthew D.; Nelson, Carl P.; Willets, Jonathon M.; Challiss, R. A. John; Davies, Noel W.
Voltage-gated potassium channels (Kv) are important regulators of membrane potential in vascular smooth muscle cells, which is integral to controlling intracellular Ca2+ concentration and regulating vascular tone. Previous work indicates that Kv channels can be modulated by receptor-driven alterations of cyclic AMP-dependent protein kinase (PKA) activity. Here, we demonstrate that Kv channel activity is maintained by tonic activity of PKA. Whole-cell recording was used to assess the effect of manipulating PKA signalling on Kv and ATP-dependent K+ channels of rat mesenteric artery smooth muscle cells. Application of PKA inhibitors, KT5720 or H89, caused a significant inhibition of Kv currents. Tonic PKA-mediated activation of Kv appears maximal as application of isoprenaline (a ?-adrenoceptor agonist) or dibutyryl-cAMP failed to enhance Kv currents. We also show that this modulation of Kv by PKA can be reversed by protein phosphatase 2B/calcineurin (PP2B). PKA-dependent inhibition of Kv by KT5720 can be abrogated by pre-treatment with the PP2B inhibitor cyclosporin A, or inclusion of a PP2B auto-inhibitory peptide in the pipette solution. Finally, we demonstrate that tonic PKA-mediated modulation of Kv requires intact caveolae. Pre-treatment of the cells with methyl-?-cyclodextrin to deplete cellular cholesterol, or adding caveolin-scaffolding domain peptide to the pipette solution to disrupt caveolae-dependent signalling each attenuated PKA-mediated modulation of the Kv current. These findings highlight a novel, caveolae-dependent, tonic modulatory role of PKA on Kv channels providing new insight into mechanisms and the potential for pharmacological manipulation of vascular tone. PMID:25793374
Dynamic properties of water around a protein-DNA complex from molecular dynamics simulations
Sudipta Kumar Sinha; Sanjoy Bandyopadhyay
Formation of protein-DNA complex is an important step in regulation of genes in living organisms. One important issue in this problem is the role played by water in mediating the protein-DNA interactions. In this work, we have carried out atomistic molecular dynamics simulations to explore the heterogeneous dynamics of water molecules present in different regions around a complex formed between
Up-regulation of hyperpolarization-activated cyclic nucleotide-gated channel 3 (HCN3) by specific interaction with K+ channel tetramerization domain-containing protein 3 (KCTD3).
Cao-Ehlker, Xiaochun; Zong, Xiangang; Hammelmann, Verena; Gruner, Christian; Fenske, Stefanie; Michalakis, Stylianos; Wahl-Schott, Christian; Biel, Martin
Most ion channels consist of the principal ion-permeating core subunit(s) and accessory proteins that are assembled with the channel core. The biological functions of the latter proteins are diverse and include the regulation of the biophysical properties of the ion channel, its connection to signaling pathways and the control of its cell surface expression. There is recent evidence that native hyperpolarization-activated cyclic nucleotide-gated channel complexes (HCN1-4) also contain accessory subunits, among which TRIP8b (tetratricopeptide repeat-containing Rab8b-interacting protein) has been most extensively studied. Here, we identify KCTD3, a so far uncharacterized member of the potassium channel tetramerization-domain containing (KCTD) protein family as an HCN3-interacting protein. KCTD3 is widely expressed in brain and some non-neuronal tissues and colocalizes with HCN3 in specific regions of the brain including hypothalamus. Within the HCN channel family, KCTD3 specifically binds to HCN3 and leads to a profound up-regulation of cell surface expression and current density of this channel. HCN3 can also functionally interact with TRIP8b; however, we found no evidence for channel complexes containing both TRIP8b and KCTD3. The C terminus of HCN3 is crucially required for functional interaction with KCTD3. Replacement of the cytosolic C terminus of HCN2 by the corresponding domain of HCN3 renders HCN2 sensitive to regulation by KCTD3. The C-terminal-half of KCTD3 is sufficient for binding to HCN3. However, the complete protein including the N-terminal tetramerization domain is needed for HCN3 current up-regulation. Together, our experiments indicate that KCTD3 is an accessory subunit of native HCN3 complexes. PMID:23382386
The A-Type Potassium Channel Kv4.2 Is a Substrate for the Mitogen-Activated Protein Kinase ERK
J. Paige Adams; Anne E. Anderson; Andrew W. Varga; Kelly T. Dineley; Richard G. Cook; Paul J. Pfaffinger; J. David Sweatt
The mitogen-activated protein kinase ERK has recently become a focus of studies of synaptic plasticity and learning and memory. Due to the prominent role of potassium channels in regulating the electrical properties of membranes, modulation of these channels by ERK could play an important role in mediating learning-related synaptic plasticity in the CNS. Kv4.2 is a Shal-type po- tassium channel
Hierarchic Theory of Condensed Matter: Role of water in protein dynamics, function and cancer emergency
Alex Kaivarainen
1. Role of inter-domain water clusters in large-scale dynamics of proteins; 2. Description of large-scale dynamics of proteins based on generalized Stokes-Einstein and Eyring-Polany equation; 3. Dynamic model of protein-ligand complexes formation; 4. The life-time of quasiparticles and frequencies of their excitation; 5. Mesoscopic mechanism of enzyme catalysis; 6. The mechanism of ATP hydrolysis energy utilization in muscle contraction and protein polymerization; 7. Water activity as a regulative factor in the intra- and inter-cell processes; 8. Water and cancer.
Dietary safety evaluation of water hyacinth leaf protein concentrate.
Wu, Wenbiao; Sun, Yanling
It has been shown that water hyacinth leaf protein concentrate (WHLPC) is nutritionally and economically available for applications in food and feed, such as biscuits or seasonings industries, but, its dietary safety has never been studied. The dietary safety of WHLPC was therefore evaluated by analyzing the contents of total alkaloids, phenolic compounds, and heavy metals, followed by laboratory animal feeding test. The total alkaloid and phenolic contents of WHLPC were 18.7 mg/kg and 5.2 mg/kg, respectively. WHLPC contained non-detectable Cd, 0.04 mg/kg Cr, 0.001 mg/kg Pb, 0.002 mg/kg Pt, 0.001 mg/kg Pd, 0.003 mg/kg Sn, 0.002 mg/kg Hg, 0.01 mg/kg Ba, 0.001 mg/kg Ag, 0.006 mg/kg Sd, and 0.03 mg/kg Al. The LD(50) of WHLPC in mice was more than 20.5 g/kg body weight (bw). After feeding mice for 7, 30, 60 or 90 days, either on diet containing WHLPC or a control diet of equivalent protein content, there were no significant differences in absolute body weight or weight gain of WHLPC-treated mice. The results of haematological analysis, histopathological evaluation, general dissection, and investigations of internal organs did not show any adverse effects from diet containing WHLPC. It was concluded that WHLPC is not acutely toxic and does not show sub-chronic in mice. PMID:21148597
Technology Transfer Automated Retrieval System (TEKTRAN)
Most research concerning grain proteins has concentrated upon the gluten storage proteins. The albumins and globulins are the water and salt soluble proteins that contain biologically active enzymes and enzyme inhibitors. A Free Zone Capillary electrophoresis method was developed to separate these p...
Coupled relaxations at the protein¢water interface in the picosecond time scale
Paciaroni, A.; Cornicchi, E.; Marconi, M.; Orecchini, A.; Petrillo, C.; Haertlein, M.; Moulin, M.; Sacchetti, F.
The spectral behaviour of a protein and its hydration water has been investigated through neutron scattering. The availability of both hydrogenated and perdeuterated samples of maltose-binding protein (MBP) allowed us to directly measure with great accuracy the signal from the protein and the hydration water alone. Both the spectra of the MBP and its hydration water show two distinct relaxations, a behaviour that is reminiscent of glassy systems. The two components have been described using a phenomenological model that includes two Cole¢Davidson functions. In MBP and its hydration water, the two relaxations take place with similar average characteristic times of approximately 10 and 0.2 ps. The common time scales of these relaxations suggest that they may be a preferential route to couple the dynamics of the water hydrogen-bond network around the protein surface with that of protein fluctuations. PMID:19640876
The 2 subunits of voltage-gated calcium channels form GPI-anchored proteins, a posttranslational
Dolphin, Annette C.
the traffick- ing and properties of CaV1 and CaV2 channels. The 2 subunits have been described as type I | electrophysiology | immunocytochemistry Voltage-gated (CaV) calcium channels of the CaV1 and CaV2 classes, Physiology and Pharmacology, University College, London WC1E 6BT, United Kingdom Edited by William A
The Influenza Virus M 2Ion Channel Protein: Probing the Structure of the Transmembrane Domain in Intact Cells by Using Engineered Disulfide Cross-Linking
Christina M. Bauer; Lawrence H. Pinto; Timothy A. Cross; Robert A. Lamb
The influenza A virus M2integral membrane protein is an ion channel that permits protons to enter virus particles during uncoating of virions in endosomes, and it also modulates the pH of thetrans-Golgi network in virus-infected cells. M2protein is a homo-oligomer of 97 residues with a single transmembrane (TM) domain whose residues encompass the pore region of the channel and the
Isoform-Specific Interaction of the alpha 1A Subunits of Brain Ca2+ Channels with the Presynaptic Proteins Syntaxin and SNAP25
Jens Rettig; Zu-Hang Sheng; D. Kyle Kim; Connie D. Hodson; Terry P. Snutch; William A. Catterall
Presynaptic Ca2+ channels are crucial elements in neuronal excitation-secretion coupling. In addition to mediating Ca2+ entry to initiate transmitter release, they are thought to interact directly with proteins of the synaptic vesicle docking\\/fusion machinery. Here we report isoform-specific, stoichiometric interaction of the BI and rbA isoforms of the alpha 1A subunit of P\\/Q-type Ca2+ channels with the presynaptic membrane proteins
Venkatachalam, Kartik; Montell, Craig
The TRP (Transient Receptor Potential) superfamily of cation channels is remarkable in that it displays greater diversity in activation mechanisms and selectivities than any other group of ion channels. The domain organizations of some TRP proteins are also unusual, as they consist of linked channel and enzyme domains. A unifying theme in this group is that TRP proteins play critical roles in sensory physiology, which include contributions to vision, taste, olfaction, hearing, touch, and thermo- and osmosensation. In addition, TRP channels enable individual cells to sense changes in their local environment. Many TRP channels are activated by a variety of different stimuli and function as signal integrators. The TRP superfamily is divided into seven subfamilies: the five group 1 TRPs (TRPC, TRPV, TRPM, TRPN, and TRPA) and two group 2 subfamilies (TRPP and TRPML). TRP channels are important for human health as mutations in at least four TRP channels underlie disease. PMID:17579562
Dynamics of laser-induced channel formation in water and influence of pulse duration on the ablation of biotissue under water with pulsed erbium-laser radiation
NASA Astrophysics Data System (ADS)
Ith, M.; Pratisto, H.; Altermatt, H. J.; Frenz, M.; Weber, H. P.
The ability to use fiber-delivered erbium-laser radiation for non-contact arthroscopic meniscectomy in a liquid environment was studied. The laser radiation is transmitted through a water-vapor channel created by the leading part of the laser pulse. The dynamics of the channel formation around a submerged fiber tip was investigated with time-resolved flash photography. Strong pressure transients with amplitudes up to a few hundreds of bars measured with a needle hydrophone were found to accompany the channel formation process. Additional pressure transients in the range of kbars were observed after the laser pulse associated with the collapse of the vapor channel. Transmission measurements revealed that the duration the laser-induced channel stays open, and therefore the energy transmittable through it, is substantially determined by the laser pulse duration. The optimum pulse duration was found to be in the range between 250 and 350 ĄS. This was confirmed by histological evaluations of the laser incisions in meniscus: Increasing the pulse duration from 300 to 800 Ąs leads to a decrease in the crater depth from 1600 to 300 Ąm. A comparison of the histological examination after laser treatment through air and through water gave information on the influence of the vapor channel on the ablation efficiency, the cutting quality and the induced thermal damage in the adjacent tissue. The study shows that the erbium laser combined with an adequate fiber delivery system represents an effective surgical instrument liable to become increasingly accepted in orthopedic surgery.
NSDL National Science Digital Library
Stacey Nee MacFarlane (University of Pennsylvania School of Medicine; Department of Neuroscience REV)
The functions of ion channels can be regulated by their phosphorylation state. Protein kinases and protein phosphatases tightly control the activity of channels, thereby regulating the flow of ions across cell membranes. Channel proteins and kinases or phosphatases can associate directly or through intermediate adaptor proteins. An interaction domain termed the leucine zipper (LZ), once thought to be unique to some families of transcription factors, has been identified in channel proteins and their cognate binding proteins. MacFarlane and Levitan discuss what roles LZ-containing proteins might have in controlling channel function.
Assembly of water-soluble chlorophyll-binding proteins with native hydrophobic chlorophylls in water-in-oil emulsions.
Bednarczyk, Dominika; Takahashi, Shigekazu; Satoh, Hiroyuki; Noy, Dror
The challenges involved in studying cofactor binding and assembly, as well as energy- and electron transfer mechanisms in the large and elaborate transmembrane protein complexes of photosynthesis and respiration have prompted considerable interest in constructing simplified model systems based on their water-soluble protein analogs. Such analogs are also promising templates and building blocks for artificial bioinspired energy conversion systems. Yet, development is limited by the challenge of introducing the essential cofactors of natural proteins that are highly water-insoluble into the water-soluble protein analogs. Here we introduce a new efficient method based on water-in-oil emulsions for overcoming this challenge. We demonstrate the effectiveness of the method in the assembly of native chlorophylls with four recombinant variants of the water-soluble chlorophyll-binding protein of Brassicaceae plants. We use the method to gain new insights into the protein-chlorophyll assembly process, and demonstrate its potential as a fast screening system for developing novel chlorophyll-protein complexes. PMID:25511505
Making water-soluble integral membrane proteins in vivo using an amphipathic protein fusion strategy
Mizrachi, Dario; Chen, Yujie; Liu, Jiayan; Peng, Hwei-Ming; Ke, Ailong; Pollack, Lois; Turner, Raymond J.; Auchus, Richard J.; DeLisa, Matthew P.
Integral membrane proteins (IMPs) play crucial roles in all cells and represent attractive pharmacological targets. However, functional and structural studies of IMPs are hindered by their hydrophobic nature and the fact that they are generally unstable following extraction from their native membrane environment using detergents. Here we devise a general strategy for in vivo solubilization of IMPs in structurally relevant conformations without the need for detergents or mutations to the IMP itself, as an alternative to extraction and in vitro solubilization. This technique, called SIMPLEx (solubilization of IMPs with high levels of expression), allows the direct expression of soluble products in living cells by simply fusing an IMP target with truncated apolipoprotein A-I, which serves as an amphipathic proteic æshield' that sequesters the IMP from water and promotes its solubilization. PMID:25851941
Making water-soluble integral membrane proteins in vivo using an amphipathic protein fusion strategy.
Mizrachi, Dario; Chen, Yujie; Liu, Jiayan; Peng, Hwei-Ming; Ke, Ailong; Pollack, Lois; Turner, Raymond J; Auchus, Richard J; DeLisa, Matthew P
Integral membrane proteins (IMPs) play crucial roles in all cells and represent attractive pharmacological targets. However, functional and structural studies of IMPs are hindered by their hydrophobic nature and the fact that they are generally unstable following extraction from their native membrane environment using detergents. Here we devise a general strategy for in vivo solubilization of IMPs in structurally relevant conformations without the need for detergents or mutations to the IMP itself, as an alternative to extraction and in vitro solubilization. This technique, called SIMPLEx (solubilization of IMPs with high levels of expression), allows the direct expression of soluble products in living cells by simply fusing an IMP target with truncated apolipoprotein A-I, which serves as an amphipathic proteic 'shield' that sequesters the IMP from water and promotes its solubilization. PMID:25851941
Management implications of the relationships between water chemistry and fishes within channelized headwater streams in the midwestern United States
Technology Transfer Automated Retrieval System (TEKTRAN)
Many headwater streams in the midwestern United States were channelized for agricultural drainage. Conservation practices are implemented to reduce nutrient and pesticide loadings within these altered streams. The impact of these practices is uncertain because the influence of water chemistry on str...
Design and simulation of a new model for shallow water multipath acoustic channel in the Persian Gulf
A. Doosti Aref; B. Abbasi Arand
In this paper, for the purpose of investigating the horizontal sound propagation, based on the results obtained from practical measurements in the Strait of Hormuz and available data on sound speed variations at different depths, a comprehensive model for shallow water multipath acoustic channel is presented. The mathematical modeling of the multipath effect is based on the ray theory and
A comparison of the value of viscosity for several water models using Poiseuille flow in a nano channel
Luding, Stefan
A comparison of the value of viscosity for several water models using Poiseuille flow in a nano & Hydrodynamics, Delft University of Technology, Leeghwaterstraat 21, 2628 CA Delft, The Netherlands and 2 Multi models SPC/E, TIP4P, TIP4P/Ew, and TIP4P/2005 by considering Poiseuille flow inside a nano channel using
Expression and isotopic labelling of the potassium channel blocker ShK toxin as a thioredoxin fusion protein in bacteria
Chang, Shih Chieh; Galea, Charles A.; Leung, Eleanor W W.; Tajhya, Rajeev B.; Beeton, Christine; Pennington, Michael W.; Norton, Raymond S.
The polypeptide toxin ShK is a potent blocker of Kv1.3 potassium channels, which play a crucial role in the activation of human effector memory T-cells (TEM). Selective blockers constitute valuable therapeutic leads for the treatment of autoimmune diseases mediated by TEM cells, such as multiple sclerosis, rheumatoid arthritis, and type-1 diabetes. We have established a recombinant peptide expression system in order to generate isotopically-labelled ShK and various ShK analogues for in-depth biophysical and pharmacological studies. ShK was expressed as a thioredoxin fusion protein in Escherichia coli BL21 (DE3) cells and purified initially by Ni2+ iminodiacetic acid affinity chromatography. The fusion protein was cleaved with enterokinase and purified to homogeneity by reverse-phase HPLC. NMR spectra of 15N-labelled ShK were similar to those reported previously for the unlabelled synthetic peptide, confirming that recombinant ShK was correctly folded. Recombinant ShK blocked Kv1.3 channels with a Kd of 25 pM and inhibited the proliferation of human and rat T lymphocytes with a preference for TEM cells, with similar potency to synthetic ShK in all assays. This expression system also enables the efficient production of 15N-labelled ShK for NMR studies of peptide dynamics and of the interaction of ShK with Kv1.3 channels. PMID:22659540
Nighttime cirrus detection using Atmospheric Infrared Sounder window channels and total column water vapor
NASA Astrophysics Data System (ADS)
Kahn, Brian H.; Liou, Kuo Nan; Lee, Sung-Yung; Fishbein, Evan F.; Desouza-Machado, Sergio; Eldering, Annmarie; Fetzer, Eric J.; Hannon, Scott E.; Strow, L. Larrabee
A method of cirrus detection at nighttime is presented that utilizes 3.8 and 10.4 ?m infrared (IR) window brightness temperature differences (dBT) and total column precipitable water (PW) measurements. This technique is applied to the Atmospheric Infrared Sounder (AIRS) and Advanced Microwave Sounding Unit A (AMSU-A) instrument suite on board EOS-Aqua, where dBT is determined from sets of carefully selected AIRS window channels, while PW is derived from the synergistic AIRS and AMSU-A water vapor retrievals. Simulated and observed dBT for a particular value of PW are not constant; several physical factors impact dBT, including the variability in temperature and relative humidity profiles, surface emissivity, instrument noise, and skin/near-surface air temperature differences. We simulate clear-sky dBT over a realistic range of PWs using 8350 radiosondes that have varying temperature and relative humidity profiles. Thresholds between cloudy and uncertain sky conditions are derived once the scatter in the clear-sky dBT is determined. Simulations of optically thin cirrus indicate that this technique is most sensitive to cirrus optical depth in the 10 ?m window of 0.1-0.15 or greater over the tropical and subtropical oceans, where surface emissivity and skin/near-surface air temperature impacts on the IR radiances are minimal. The method at present is generally valid over oceanic regions only, specifically, the tropics and subtropics. The detection of thin cirrus, and other cloud types, is validated using observations at the Atmospheric Radiation Measurement (ARM) program site located at Manus Island in the tropical western Pacific for 89 coincident EOS-Aqua overpasses. Even though the emphasis of this work is on the detection of thin cirrus at nighttime, this technique is sensitive to a broad cloud morphology. The cloud detection technique agrees with ARM-detected clouds 82-84% of the time, which include thin cirrus, as well as other cloud types. Most of the disagreements are well explained by AIRS footprint-scale heterogeneity compared to ARM point measurements, cirrus overlying lower-layer water clouds, possible mixed phase microphysics in midlevel clouds, and significant IR channel noise for cold BT scenes over deep convective towers.
Nighttime Cirrus Detection using Atmospheric Infrared Sounder Window Channels and Total Column Water Vapor
NASA Technical Reports Server (NTRS)
Kahn, Brian H.; Liou, Kuo Nan; Lee, Sung-Yung; Fishbein, Evan F.; DeSouza-Machado, Sergio; Eldering, Annmarie; Fetzer, Eric J.; Hannon, Scott E.; Strow, L. Larrabee
A method of cirrus detection at nighttime is presented that utilizes 3.8 and 10.4 (micro)m infrared (IR) window brightness temperature differences (dBT) and total column precipitable water (PW) measurements. This technique is applied to the Atmospheric Infrared Sounder (AIRS) and Advanced Microwave Sounding Unit A (AMSU-A) instrument suite on board EOS-Aqua, where dBT is determined from sets of carefully selected AIRS window channels, while PW is derived from the synergistic AIRS and AMSU-A water vapor retrievals. Simulated and observed dBT for a particular value of PW are not constant; several physical factors impact dBT, including the variability in temperature and relative humidity profiles, surface emissivity, instrument noise, and skin/ near-surface air temperature differences. We simulate clear-sky dBT over a realistic range of PWs using 8350 radiosondes that have varying temperature and relative humidity profiles. Thresholds between cloudy and uncertain sky conditions are derived once the scatter in the clear-sky dBT is determined. Simulations of optically thin cirrus indicate that this technique is most sensitive to cirrus optical depth in the 10 (micro)m window of 0.1-0.15 or greater over the tropical and subtropical oceans, where surface emissivity and skin/near-surface air temperature impacts on the IR radiances are minimal. The method at present is generally valid over oceanic regions only, specifically, the tropics and subtropics. The detection of thin cirrus, and other cloud types, is validated using observations at the Atmospheric Radiation Measurement (ARM) program site located at Manus Island in the tropical western Pacific for 89 coincident EOS-Aqua overpasses. Even though the emphasis of this work is on the detection of thin cirrus at nighttime, this technique is sensitive to a broad cloud morphology. The cloud detection technique agrees with ARM-detected clouds 82-84% of the time, which include thin cirrus, as well as other cloud types. Most of the disagreements are well explained by AIRS footprint-scale heterogeneity compared to ARM point measurements, cirrus overlying lower-layer water clouds, possible mixed phase microphysics in midlevel clouds, and significant IR channel noise for cold BT scenes over deep convective towers.
Molecular Dynamics Simulations of Proteins: Can the Explicit Water Model Be Varied?
Smith, Jeremy C [ORNL; Nutt, David [University of Heidelberg
In molecular mechanics simulations of biological systems, the solvation water is typically represented by a default water model which is an integral part of the force field. Indeed, protein nonbonding parameters are chosen in order to obtain a balance between water-water and protein-water interactions and hence a reliable description of protein solvation. However, less attention has been paid to the question of whether the water model provides a reliable description of the water properties under the chosen simulation conditions, for which more accurate water models often exist. Here we consider the case of the CHARMM protein force field, which was parameterized for use with a modified TIP3P model. Using quantum mechanical and molecular mechanical calculations, we investigate whether the CHARMM force field can be used with other water models: TIP4P and TIP5P. Solvation properties of N-methylacetamide (NMA), other small solute molecules, and a small protein are examined. The results indicate differences in binding energies and minimum energy geometries, especially for TIP5P, but the overall description of solvation is found to be similar for all models tested. The results provide an indication that molecular mechanics simulations with the CHARMM force field can be performed with water models other than TIP3P, thus enabling an improved description of the solvent water properties.
Molecular dynamics simulations of proteins: can the explicit water model be varied?
Nutt, David [University of Heidelberg; Smith, Jeremy C [ORNL
In molecular mechanics simulations of biological systems, the solvation water is typically represented by a default water model which is an integral part of the force field. Indeed, protein nonbonding parameters are chosen in order to obtain a balance between water-water and protein-water interactions and hence a reliable description of protein solvation. However, less attention has been paid to the question of whether the water model provides a reliable description of the water properties under the chosen simulation conditions, for which more accurate water models often exist. Here we consider the case of the CHARMM protein force field, which was parameterized for use with a modified TIP3P model. Using quantum mechanical and molecular mechanical calculations, we investigate whether the CHARMM force field can be used with other water models: TIP4P and TIP5P. Solvation properties of N-methylacetamide (NMA), other small solute molecules, and a small protein are examined. The results indicate differences in binding energies and minimum energy geometries, especially for TIP5P, but the overall description of solvation is found to be similar for all models tested. The results provide an indication that molecular mechanics simulations with the CHARMM force field can be performed with water models other than TIP3P, thus enabling an improved description of the solvent water properties.
HYDRODYNAMICS AND OXYGEN MODELING OF THE STOCKTON DEEP WATER SHIP CHANNEL i HYDRODYNAMICS AND OXYGEN MODELING OF THE STOCKTON DEEP WATER
Schladow, S. Geoffrey
WATER SHIP CHANNEL iv downstream estuarine temperatures. The thermal energy balance suggested occurred in the past. Each experiment consisted of a 3-5 week deployment of acoustic Doppler current showed that a balance of advection, dispersion and surface forcing determines sub-tidal temperature
Anisotropic dynamics of water ultraconfined in macroscopically oriented channels of single-crystal beryl: A multifrequency analysis
NASA Astrophysics Data System (ADS)
Anovitz, Lawrence M.; Mamontov, Eugene; ben Ishai, Paul; Kolesnikov, Alexander I.
The properties of fluids can be significantly altered by the geometry of their confining environments. While there has been significant work on the properties of such confined fluids, the properties of fluids under ultraconfinement, environments where, at least in one plane, the dimensions of the confining environment are similar to that of the confined molecule, have not been investigated. This paper investigates the dynamic properties of water in beryl (Be3Al2Si6O18), the structure of which contains approximately 5-┼-diam channels parallel to the c axis. Three techniques, inelastic neutron scattering, quasielastic neutron scattering, and dielectric spectroscopy, have been used to quantify these properties over a dynamic range covering approximately 16 orders of magnitude. Because beryl can be obtained in large single crystals we were able to quantify directional variations, perpendicular and parallel to the channel directions, in the dynamics of the confined fluid. These are significantly anisotropic and, somewhat counterintuitively, show that vibrations parallel to the c-axis channels are significantly more hindered than those perpendicular to the channels. The effective potential for vibrations in the c direction is harder than the potential in directions perpendicular to it. There is evidence of single-file diffusion of water molecules along the channels at higher temperatures, but below 150 K this diffusion is strongly suppressed. No such suppression, however, has been observed in the channel-perpendicular direction. Inelastic neutron scattering spectra include an intramolecular stretching O-H peak at ś465 meV. As this is nearly coincident with that known for free water molecules and approximately 30 meV higher than that in liquid water or ice, this suggests that there is no hydrogen bonding constraining vibrations between the channel water and the beryl structure. However, dielectric spectroscopic measurements at higher temperatures and lower frequencies yield an activation energy for the dipole reorientation of 16.4 ▒ 0.14 kJ/mol, close to the energy required to break a hydrogen bond in bulk water. This may suggest the presence of some other form of bonding between the water molecules and the structure, but the resolution of the apparent contradiction between the inelastic neutron and dielectric spectroscopic results remains uncertain.
The role of water exchange between a stream channel and its hyporheic zone in nitrogen cycling at the terrestrial-aquatic interface
Frank J. Triska; John H. Duff; Ronald J. Avanzino
The subsurface riparian zone was examined as an ecotone with two interfaces. Inland is a terrestrial boundary, where transport of water and dissolved solutes is toward the channel and controlled by watershed hydrology. Streamside is an aquatic boundary, where exchange of surface water and dissolved solutes is bi-directional and flux is strongly influenced by channel hydraulics. Streamside, bi-directional exchange of
A New Criterion to Evaluate Water Vapor Interference in Protein Secondary Structural Analysis by FTIR Spectroscopy
Zou, Ye; Ma, Gang
Second derivative and Fourier self-deconvolution (FSD) are two commonly used techniques to resolve the overlapped component peaks from the often featureless amide I band in Fourier transform infrared (FTIR) curve-fitting approach for protein secondary structural analysis. Yet, the reliability of these two techniques is greatly affected by the omnipresent water vapor in the atmosphere. Several criteria are currently in use as quality controls to ensure the protein absorption spectrum is negligibly affected by water vapor interference. In this study, through a second derivative study of liquid water, we first argue that the previously established criteria cannot guarantee a reliable evaluation of water vapor interference due to a phenomenon that we refer to as sampleÆs absorbance-dependent water vapor interference. Then, through a comparative study of protein and liquid water, we show that a protein absorption spectrum can still be significantly affected by water vapor interference even though it satisfies the established criteria. At last, we propose to use the comparison between the second derivative spectra of protein and liquid water as a new criterion to better evaluate water vapor interference for more reliable second derivative and FSD treatments on the protein amide I band. PMID:24901531
Golgi Anti-apoptotic Proteins Are Highly Conserved Ion Channels That Affect Apoptosis and Cell Migration
Carrara, Guia; Saraiva, Nuno; Parsons, Maddy; Byrne, Bernadette; Prole, David L.; Taylor, Colin W.; Smith, Geoffrey L.
Golgi anti-apoptotic proteins (GAAPs) are multitransmembrane proteins that are expressed in the Golgi apparatus and are able to homo-oligomerize. They are highly conserved throughout eukaryotes and are present in some prokaryotes and orthopoxviruses...
the dynamics of water molecules bound to proteins to be unambiguously resolved for the
Tennessee, University of
. In other words, the geometric con- straintsimposedbyubiquitinonthesurround- ing water molecules effectivelythe dynamics of water molecules bound to proteins to be unambiguously resolved for the first time. The authors' key finding is that water molecules that have similar hydration dynam- ics form clusters across
PoreWalker: A Novel Tool for the Identification and Characterization of Channels in Transmembrane Proteins from Their Three-Dimensional Structure
Thornton, Janet M.
Transmembrane channel proteins play pivotal roles in maintaining the homeostasis and responsiveness of cells and the cross-membrane electrochemical gradient by mediating the transport of ions and molecules through biological membranes. Therefore, computational methods which, given a set of 3D coordinates, can automatically identify and describe channels in transmembrane proteins are key tools to provide insights into how they function. Herein we present PoreWalker, a fully automated method, which detects and fully characterises channels in transmembrane proteins from their 3D structures. A stepwise procedure is followed in which the pore centre and pore axis are first identified and optimised using geometric criteria, and then the biggest and longest cavity through the channel is detected. Finally, pore features, including diameter profiles, pore-lining residues, size, shape and regularity of the pore are calculated, providing a quantitative and visual characterization of the channel. To illustrate the use of this tool, the method was applied to several structures of transmembrane channel proteins and was able to identify shape/size/residue features representative of specific channel families. The software is available as a web-based resource at http://www.ebi.ac.uk/thornton-srv/software/PoreWalker/. PMID:19609355
The grapevine tonoplast aquaporin TIP2;1 is a pressure gated water channel.
LeitŃo, LuĒs; Prista, Catarina; Loureiro-Dias, Maria C; Moura, Teresa F; Soveral, Grańa
In plants, the vacuole is a multifunctional organelle with an important role in the maintenance of the intracellular space. Tonoplast membranes are highly permeable to water due to their content in aquaporins TIPs (Tonoplast Intrinsic Proteins) that allow the rapid water influx creating an internal turgor pressure responsible for cell expansion, elongation and shape. The aim of the present study was to evaluate if the grapevine Vitis vinifera TIP2;1 would operate as a possible volume regulator gated by membrane surface tension. For that, the wild type VvTIP2;1 and a non-functional mutated form were heterologous expressed in yeast. Using an experimental strategy in which cells are incubated in external media that induce an increase in internal hydrostatic pressure and consequently membrane surface tension, we were able to compare the osmotic permeability (Pf) and the activation energy for water transport (Ea) of yeast strains expressing the functional and a non-functional TIP2;1. We found Pf and Ea dependence on internal turgor pressure only for the strain harboring the functional aquaporin indicating that TIP2;1 activity is regulated by membrane tension changing from an open to a closed state in an internal pressure dependent manner. This turgor dependent gating of TIP2;1 might be a mechanism to regulate vacuolar size and shape in plants withstanding hostile drought conditions such as grapevine. PMID:24942877
Structure and Inhibition of the SARS Coronavirus Envelope Protein Ion Channel
Konstantin Pervushin; Edward Tan; Krupakar Parthasarathy; Xin Lin; Feng Li Jiang; Dejie Yu; Ardcharaporn Vararattanavech; Tuck Wah Soong; Ding Xiang Liu; Jaume Torres
The envelope (E) protein from coronaviruses is a small polypeptide that contains at least one ?-helical transmembrane domain. Absence, or inactivation, of E protein results in attenuated viruses, due to alterations in either virion morphology or tropism. Apart from its morphogenetic properties, protein E has been reported to have membrane permeabilizing activity. Further, the drug hexamethylene amiloride (HMA), but not
Silver nanoparticle exposure induces rat motor dysfunction through decrease in expression of calcium channel protein in cerebellum.
Yin, Nuoya; Zhang, Yang; Yun, Zhaojun; Liu, Qian; Qu, Guangbo; Zhou, Qunfang; Hu, Ligang; Jiang, Guibin
Silver nanoparticles (AgNPs) are currently used widely, however, their impact on central nervous system still remains ambiguous and needs to be elucidated. This study is performed to investigate the neurotoxicity of AgNPs and illustrate the potential molecular mechanism. Neonatal Sprague-Dawley (SD) rats are exposed to AgNPs by intranasal instillation for 14 weeks. It is demonstrated that AgNPs exposure causes cerebellar ataxia like symptom in rats, evidenced by dysfunction of motor coordination and impairment of locomotor activity. Observation of cerebellum section reveals that AgNPs can provoke destruction of cerebellum granular layer with concomitant activation of glial cells. AgNPs treatment decreases calcium channel protein (CACNA1A) levels in cerebellum without changing potassium channel protein (KCNA1) levels. The levels of silver in rat cerebellum tissue are correlated with exposure doses. In vitro experiments reveal that AgNPs treatment significantly reduces the protein and mRNA levels of CACNA1A in primary cultured cerebellum granule cells (CGCs). These findings suggest that AgNPs-induced rat motor dysfunction is associated with CACNA1A expression decrease, which reveals the underlying molecular mechanism for the neurotoxicity of AgNPs. Possible counteractions may accordingly be suggested to attenuate the unexpected harmful effects in biological applications of AgNPs. PMID:26068065
Structural characterization of the voltage sensor domain and voltage-gated K+- channel proteins vectorially-oriented within a single bilayer membrane at the solid/vapor and solid/liquid interfaces via neutron interferometry
Gupta, S.; Dura, J.A.; Freites, J.A.; Tobias, D.J.; Blasie, J. K.
The voltage-sensor domain (VSD) is a modular 4-helix bundle component that confers voltage sensitivity to voltage-gated cation channels in biological membranes. Despite extensive biophysical studies and the recent availability of x-ray crystal structures for a few voltage-gated potassium (Kv-) channels and a voltage-gate sodium (Nav-) channel, a complete understanding of the cooperative mechanism of electromechanical coupling, interconverting the closed-to-open states (i.e. non-conducting to cation conducting) remains undetermined. Moreover, the function of these domains is highly dependent on the physical-chemical properties of the surrounding lipid membrane environment. The basis for this work was provided by a recent structural study of the VSD from a prokaryotic Kv-channel vectorially-oriented within a single phospholipid (POPC; 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) membrane investigated by x-ray interferometry at the solid/moist He (or solid/vapor) and solid/liquid interfaces thus achieving partial to full hydration, respectively (Gupta et. al. Phys. Rev E. 2011, 84). Here, we utilize neutron interferometry to characterize this system in substantially greater structural detail at the sub-molecular level, due to its inherent advantages arising from solvent contrast variation coupled with the deuteration of selected sub-molecular membrane components, especially important for the membrane at the solid/liquid interface. We demonstrate the unique vectorial orientation of the VSD and the retention of its molecular conformation manifest in the asymmetric profile structure of the protein within the profile structure of this single bilayer membrane system. We definitively characterize the asymmetric phospholipid bilayer solvating the lateral surfaces of the VSD protein within the membrane. The profile structures of both the VSD protein and phospholipid bilayer depend upon the hydration state of the membrane. We also determine the distribution of water and exchangeable hydrogen throughout the profile structure of both the VSD itself and the VSD:POPC membrane. These two experimentally-determined water and exchangeable hydrogen distribution profiles are in good agreement with molecular dynamics simulations of the VSD protein vectorially-oriented within a fully hydrated POPC bilayer membrane, supporting the existence of the VSDÆs water pore. This approach was extended to the full-length Kv-channel (KvAP) at solid/liquid interface, providing the separate profile structures of the KvAP protein and the POPC bilayer within the reconstituted KvAP:POPC membrane. PMID:22686684
Tissue-specific distribution and variation of the channel-forming protein ductin during development of Drosophila melanogaster.
Bohrmann, J; Bonafede, A
Ductins represent membrane channel proteins which are supposed to form both proton channels in V-ATPases and connexon channels in gap junctions. In order to localize and characterize these proteins in different tissues of Drosophila, we applied indirect immunofluorescence microscopy and immunoblots, using antisera prepared against Drosophila ductin and against Nephrops ductin. Previously, these antisera have been shown to recognize, in ovarian follicles and young embryos of Drosophila, the ductin monomer of 16 kDa and a putative dimer of 29 kDa. Moreover, both anti-ductin sera label antigens in plasma membranes and in the cytoplasm and block, when microinjected, cell-cell communication via gap junctions. In the present study, comparing several embryonic, larval and adult tissues, the anti-ductin sera were found to recognize antigens with various locations in cells of the midgut, the salivary gland, the nervous system, the muscles and the epidermis. For example, in midgut cells, antigens were labeled mainly in apical plasma membranes and in the apical part of the cytoplasm, while in salivary-gland cells, labeling was found throughout the plasma membranes and the cytoplasm. We conclude that putative gap junctions were revealed in the salivary gland, the nervous system and the epidermis, while plasma membrane-associated putative V-ATPases were detected in the midgut, the salivary gland and the muscles. Moreover, V-ATPases associated with cytoplasmic vesicles were found in almost every tissue. On immunoblots of homogenates from various tissues, the anti-ductin sera specifically labeled bands of 16, 21 and 29 kDa. When comparing these bands using peptide mapping with V8 protease, we found that they represent closely related proteins. Therefore, either different ductins or modifications of a single ductin appear to be present in different cellular regions, cell types and developmental stages of Drosophila. PMID:11206329
Denatured Mammalian Protein Mixtures Exhibit Unusually High Solubility in Nucleic Acid-Free Pure Water
Futami, Junichiro; Fujiyama, Haruna; Kinoshita, Rie; Nonomura, Hidenori; Honjo, Tomoko; Tada, Hiroko; Matsushita, Hirokazu; Abe, Yoshito; Kakimi, Kazuhiro
Preventing protein aggregation is a major goal of biotechnology. Since protein aggregates are mainly comprised of unfolded proteins, protecting against denaturation is likely to assist solubility in an aqueous medium. Contrary to this concept, we found denatured total cellular protein mixture from mammalian cell kept high solubility in pure water when the mixture was nucleic acids free. The lysates were prepared from total cellular protein pellet extracted by using guanidinium thiocyanate-phenol-chloroform mixture of TRIzol, denatured and reduced total protein mixtures remained soluble after extensive dialysis against pure water. The total cell protein lysates contained fully disordered proteins that readily formed large aggregates upon contact with nucleic acids or salts. These findings suggested that the highly flexible mixtures of disordered proteins, which have fully ionized side chains, are protected against aggregation. Interestingly, this unusual solubility is characteristic of protein mixtures from higher eukaryotes, whereas most prokaryotic protein mixtures were aggregated under identical conditions. This unusual solubility of unfolded protein mixtures could have implications for the study of intrinsically disordered proteins in a variety of cells. PMID:25405999
Gestational Hypoxia Up-regulates Protein Kinase C and Inhibits Calcium-Activated Potassium Channels in Ovine Uterine Arteries
Xiao, Daliao; Zhu, Ronghui; Zhang, Lubo
Objective: The present study tested the hypothesis that gestational hypoxia up-regulates protein kinase C (PKC) and inhibits calcium-activated potassium channels (KCa)-mediated relaxations of uterine arteries in pregnancy. Study design: Uterine arteries were isolated from nonpregnant (NPUA) and pregnant (PUA) (~140 day gestation) sheep maintained at either sea level or high altitude (3,820 m for 110 days, PaO2: 60 mmHg). Contractions of uterine arteries were determined. Key findings: In normoxic PUA, selective inhibition of large-conductance KCa (BK) channels significantly enhanced PKC activator phorbol 12, 13-dibutyrate (PDBu)-induced contractions. This effect was abrogated by chronic hypoxia in gestation. Unlike BK channels, inhibition of small-conductance KCa (SK) channels had no significant effect on PDBu-mediated contractions. In normoxic PUA, activation of both BK with NS1619 or SK with NS309 produced concentration-dependent relaxations, which were not altered by the addition of PDBu. However, in uterine arteries treated with chronic hypoxia (10.5% O2 for 48 h), both NS1619- and NS309-induced relaxations were significantly attenuated by PDBu. In NPUAs, inhibition of BK channels significantly enhanced PDBu-induced contractions in both normoxic and hypoxic animals. Conclusion: The results suggest that in the normoxic condition BK inhibits PKC activity and uterine vascular contractility, which is selectively attenuated by chronic hypoxia during gestation. In addition, hypoxia induces PKC-mediated inhibition of BK and SK activities and relaxations of uterine arteries in pregnancy. PMID:25013368
Maximizing recovery of water-soluble proteins through acetone precipitation.
Crowell, Andrew M J; Wall, Mark J; Doucette, Alan A
Solvent precipitation is commonly used to purify protein samples, as seen with the removal of sodium dodecyl sulfate through acetone precipitation. However, in its current practice, protein loss is believed to be an inevitable consequence of acetone precipitation. We herein provide an in depth characterization of protein recovery through acetone precipitation. In 80% acetone, the precipitation efficiency for six of 10 protein standards was poor (ca. ?15%). Poor recovery was also observed for proteome extracts, including bacterial and mammalian cells. As shown in this work, increasing the ionic strength of the solution dramatically improves the precipitation efficiency of individual proteins, and proteome mixtures (ca. 80-100% yield). This is obtained by including 1-30 mM NaCl, together with acetone (50-80%) which maximizes protein precipitation efficiency. The amount of salt required to restore the recovery correlates with the amount of protein in the sample, as well as the intrinsic protein charge, and the dielectric strength of the solution. This synergistic approach to protein precipitation in acetone with salt is consistent with a model of ion pairing in organic solvent, and establishes an improved method to recover proteins and proteome mixtures in high yield. PMID:24016582
Initiation of a discharge channel in water by means of electrical explosion of aluminum foil
NASA Astrophysics Data System (ADS)
Sil'nikov, M. V.; Krivosheev, S. I.; Kulakov, K. S.; Kulakov, S. L.
This paper reports the results of an experimental investigation into initiation of the electric discharge in service water by means of explosion of aluminum foil having various mass and dimensions. The electric discharge was formed in a chamber with a movable wall (the piston). As an electric energy storage, the capacitor bank having the capacity C = 200-600 ?F with charging voltage U 0 = 2-5 kV (stored energy Q 0 = 0.4-7.5 kJ) and the rate of rise of the discharging current dI/ dt = (3-4) ū 109 A/s. The results of experiments showed that destruction (loss of conductivity) of foil occurs at the value of the integral of the current density h j = (0.3-0.65) ū 109 (A2/cm4)/s. The stage of the repeated breakdown in the electric discharge occurs when the value of the intensity of the electric field along the discharge channel is of E rb ? 50 V/mm. Geometric dimensions and mass of the initiating conductor that provide the maximum efficiency of conversion of the value of Q 0 into kinetic energy of the piston have been determined.
Antimicrobial activity in the skin of the channel catfish Ictalurus punctatus: characterization of broad-spectrum histone-like antimicrobial proteins
D. Robinette; S. Wada; T. Arroll; M. G. Levy; W. L. Miller; E. J. Noga
.ĀĀ Three antibacterial proteins were isolated from acid extracts of channel catfish (Ictalurus punctatus) skin by cation exchange chromatography and reverse-phase high-pressure liquid chromatography. The molecular masses of the\\u000a proteins were 15.5, 15.5 and 30 kD as determined by SDS-polyacrylamide gel electrophoresis. Mass spectrometry, amino acid\\u000a composition and amino acid sequence data suggest that the most abundant protein is closely
Properties of channels reconstituted from the major intrinsic protein of lens fiber membranes
Detergent-solubilized plasma membrane protein of either adult bo- vine or calf lens and high-performance liquid chromatography-purified major intrinsic protein (MIP) of the lens were reconstituted into unilamellar vesicles and planar lipid bilayers. Freeze-fracture studies showed that the density of intramem- brane panicles in the vesicles was proportional to the protein\\/lipid ratio. At high ratios, these particles crystallized into tetragonal arrays
Intrinsically disordered proteins aggregate at fungal cell-to-cell channels and regulate
Wong, Limsoon
that identifies related proteins based on composition and character, we identify 17 septal pore-associated (SPA organization | filamentous fungus | Neurospora crassa Two distinct strategies provide the foundation
Alzheimer's disease amyloid beta-protein forms Zn(2+)-sensitive, cation-selective channels across excised membrane patches from hypothalamic neurons.
Kawahara, M; Arispe, N; Kuroda, Y; Rojas, E
We have previously shown that the 40-residue peptide termed amyloid beta-protein (A beta P[1-40]) in solution forms cation-selective channels across artificial phospholipid bilayer membranes. To determine whether A beta P[1-40] also forms channels across natural membranes, we used electrically silent excised membrane patches from a cell line derived from hypothalamic gonadotrophin-releasing hormone GnRH neurons. We found that exposing either the internal or the external side of excised membrane patches to A beta P[1-40] leads to the spontaneous formation of cation-selective channels. With Cs+ as the main cation in both the external as well as the internal saline, the amplitude of the A beta P[1-40] channel currents was found to follow the Cs+ gradient and to exhibit spontaneous conductance changes over a wide range (50-500 pS). We also found that free zinc (Zn2+), reported to bind to amyloid beta-protein in solution, can block the flow of Cs+ through the A beta P[1-40] channel. Because the Zn2+ chelator o-phenanthroline can reverse this blockade, we conclude that the underlying mechanism involves a direct interaction between the transition element Zn2+ and sites in the A beta P[1-40] channel pore. These properties of the A beta P[1-40] channel are rather similar to those observed in the artificial bilayer system. We also show here, by immunocytochemical confocal microscopy, that amyloid beta-protein molecules form deposits closely associated with the plasma membrane of a substantial fraction of the GnRH neurons. Taken together, these results suggest that the interactions between amyloid beta-protein and neuronal membranes also occur in vivo, lending further support to the idea that A beta P[1-40] channel formation might be a mechanism of amyloid beta-protein neurotoxicity. Images FIGURE 5 PMID:9199772
Restricted dynamics of water around a protein-carbohydrate complex: computer simulation studies.
Jana, Madhurima; Bandyopadhyay, Sanjoy
Water-mediated protein-carbohydrate interaction is a complex phenomenon responsible for different biological processes in cellular environment. One of the unexplored but important issues in this area is the role played by water during the recognition process and also in controlling the microscopic properties of the complex. In this study, we have carried out atomistic molecular dynamics simulations of a protein-carbohydrate complex formed between the hyaluronan binding domain of the murine Cd44 protein and the octasaccharide hyaluronan in explicit water. Efforts have been made to explore the heterogeneous influence of the complex on the dynamic properties of water present in different regions around it. It is revealed from our analyses that the heterogeneous dynamics of water around the complex are coupled with differential time scales of formation and breaking of hydrogen bonds at the interface. Presence of a highly rigid thin layer of motionally restricted water molecules bridging the protein and the carbohydrate in the common region of the complex has been identified. Such water molecules are expected to play a crucial role in controlling properties of the complex. Importantly, it is demonstrated that the formation of the protein-carbohydrate complex affects the transverse and longitudinal degrees of freedom of the interfacial water molecules in a heterogeneous manner. PMID:22894384
The cGMP-dependent protein kinase II Is an inhibitory modulator of the hyperpolarization-activated HCN2 channel.
Hammelmann, Verena; Zong, Xiangang; Hofmann, Franz; Michalakis, Stylianos; Biel, Martin
Opening of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels is facilitated by direct binding of cyclic nucleotides to a cyclic nucleotide-binding domain (CNBD) in the C-terminus. Here, we show for the first time that in the HCN2 channel cGMP can also exert an inhibitory effect on gating via cGMP-dependent protein kinase II (cGKII)-mediated phosphorylation. Using coimmunoprecipitation and immunohistochemistry we demonstrate that cGKII and HCN2 interact and colocalize with each other upon heterologous expression as well as in native mouse brain. We identify the proximal C-terminus of HCN2 as binding region of cGKII and show that cGKII phosphorylates HCN2 at a specific serine residue (S641) in the C-terminal end of the CNBD. The cGKII shifts the voltage-dependence of HCN2 activation to 2-5 mV more negative voltages and, hence, counteracts the stimulatory effect of cGMP on gating. The inhibitory cGMP effect can be either abolished by mutation of the phosphorylation site in HCN2 or by impairing the catalytic domain of cGKII. By contrast, the inhibitory effect is preserved in a HCN2 mutant carrying a CNBD deficient for cGMP binding. Our data suggest that bidirectional regulation of HCN2 gating by cGMP contributes to cellular fine-tuning of HCN channel activity. PMID:21347269
Importance of Translational Entropy of Water in Biological Self-Assembly Processes like Protein Folding
Kinoshita, Masahiro
We briefly review our studies on the folding/unfolding mechanisms of proteins. In biological self-assembly processes such as protein folding, the number of accessible translational configurations of water in the system increases greatly, leading to a large gain in the water entropy. The usual view looking at only the water in the close vicinity of the protein surface is capable of elucidating neither the large entropic gain upon apoplastocyanin folding, which has recently been found in a novel experimental study, nor the pressure and cold denaturation. With the emphasis on the translational entropy of water, we are presently constructing a reliable method for predicting the native structure of a protein from its amino-acid sequence. PMID:19399238
Neutron scattering evidence of a boson peak in protein hydration water Alessandro Paciaroni,1
Tuscia, UniversitÓ Degli Studi Della
Neutron scattering evidence of a boson peak in protein hydration water Alessandro Paciaroni,1 Anna, has been detected by neutron scattering and Raman spectros- copy in a large variety of glassy systems
Dynamics of a globular protein and its hydration water studied by neutron scattering and MD simulations
Chen, Sow-Hsin
This review article describes our neutron scattering experiments made in the past four years for the understanding of the single-particle (hydrogen atom) dynamics of a protein and its hydration water and the strong coupling ...
Observation of ice-like water layers at an aqueous protein surface
Meister, Konrad; Strazdaite, Simona; DeVries, Arthur L.; Lotze, Stephan; Olijve, Luuk L. C.; Voets, Ilja K.; Bakker, Huib J.
We study the properties of water at the surface of an antifreeze protein with femtosecond surface sum frequency generation spectroscopy. We find clear evidence for the presence of ice-like water layers at the ice-binding site of the protein in aqueous solution at temperatures above the freezing point. Decreasing the temperature to the biological working temperature of the protein (0 ░C to ?2 ░C) increases the amount of ice-like water, while a single point mutation in the ice-binding site is observed to completely disrupt the ice-like character and to eliminate antifreeze activity. Our observations indicate that not the protein itself but ordered ice-like water layers are responsible for the recognition and binding to ice. PMID:25468976
Observation of ice-like water layers at an aqueous protein surface.
Meister, Konrad; Strazdaite, Simona; DeVries, Arthur L; Lotze, Stephan; Olijve, Luuk L C; Voets, Ilja K; Bakker, Huib J
We study the properties of water at the surface of an antifreeze protein with femtosecond surface sum frequency generation spectroscopy. We find clear evidence for the presence of ice-like water layers at the ice-binding site of the protein in aqueous solution at temperatures above the freezing point. Decreasing the temperature to the biological working temperature of the protein (0 ░C to -2 ░C) increases the amount of ice-like water, while a single point mutation in the ice-binding site is observed to completely disrupt the ice-like character and to eliminate antifreeze activity. Our observations indicate that not the protein itself but ordered ice-like water layers are responsible for the recognition and binding to ice. PMID:25468976
Physicochemical effects of temperature and water chemistry on cohesive channel erosion
NASA Astrophysics Data System (ADS)
Wynn-Thompson, T.; Hoomehr, S.; Parks, O.; Eick, M.
One potential unforeseen consequence urbanization and climate change is accelerated stream channel erosion due to increased stream temperatures and changes in stream chemistry, which affect the surface potential and hence the stability of soil colloids. Summer thunderstorms in urban watersheds can increase stream temperature more than 7 degC and the impact of global warming on average stream temperature is already evident in some stream systems. The goal of this research was to evaluate the impact of changes in stream chemistry commonly observed in urban watersheds, and expected to occur due to climate change, on the fluvial erosion of cohesive streambank soils. We hypothesized that increases in stream temperature and changes in stream pH and salt concentrations alter the surface potential of clay particles, affecting soil erodibility. We tested this hypothesis by measuring the erosion rate of two riparian soils dominated by different common phyllosilicate clays in a recirculating hydraulic flume. Two pH levels (6, 8), three water temperatures (10 degC, 20 degC, 30 degC), and two NaCl concentrations (5 mg/l, 5 g/l) were analyzed. Velocity profiles and the distance to the soil sample were measured using a Sontek Vectrino II acoustic Doppler profiler. Additionally, zetapotential was measured to determine if erosion rates were correlated to changes in clay surface potential due to varying water chemistry. Initial study results indicated significant increases in erosion rates for both clay types with decreasing pH and increasing water temperature; temperature effects were more significant than pH effects. Changes in erosion rates with salt concentration were only significant for the soil with montmorillonite clay. While the research is ongoing, these initial results could have wide-ranging implications for climate change and urban stormwater management. Assuming climate change will result in higher stream temperatures and lower stream pH, streambank erosion could accelerate in currently stable stream systems with cohesive banks. Additionally, new stormwater management and urban design techniques may be needed to manage runoff temperature and pH, in addition to current techniques for reducing runoff peak flow and volume from developed lands.
Retrieval of Temperature and Water Vapour From Multiple Channel Lidar Systems Using an Optimal Estimation Method
NASA Astrophysics Data System (ADS)
Sica, Robert; Haefele, Alexander
While the application of optimal estimation methods (OEMs) is well-known for the retrieval of atmospheric parameters from passive instruments, active instruments have typically not employed the OEM. For instance, the measurement of temperature in the middle atmosphere with Rayleigh-scatter lidars is an important technique for assessing atmospheric change. Current retrieval schemes for these temperatures have several shortcomings which can be overcome using an OEM. Forward models have been constructed that fully characterize the measurement and allow the simultaneous retrieval of temperature, dead time and background. The OEM allows a full uncertainty budget to be obtained on a per profile basis that includes, in addition to the statistical uncertainties, the smoothing error and uncertainties due to Rayleigh extinction, ozone absorption, the lidar constant, nonlinearity in the counting system, variation of the Rayleigh-scatter cross section with altitude, pressure, acceleration due to gravity and the variation of mean molecular mass with altitude. The vertical resolution of the temperature profile is found at each height, and a quantitative determination is made of the maximum height to which the retrieval is valid. A single temperature profile can be retrieved from measurements with multiple channels that cover different height ranges, vertical resolutions and even different detection methods. The OEM employed is shown to give robust estimates of temperature consistent with previous methods, while requiring minimal computational time. Retrieval of water vapour mixing ratio from vibrational Raman scattering lidar measurements is another example where an OEM offers a considerable advantage over the standard analysis technique, with the same advantages as discussed above for Rayleigh-scatter temperatures but with an additional benefit. The conversion of the lidar measurement into mixing ratio requires a calibration constant to be employed. Using OEM the calibration constant can be retrieved if additional water vapour measurements, such as those provided by a radiosonde or microwave radiometer, are included. The success of lidar temperature and composition retrievals using an OEM opens new possibilities in atmospheric science for measurement integration between active and passive remote sensing instruments. This presentation will highlight some of these possibilities, as well as show temperature and water vapour retrievals from the MķtķoSuisse Raman Lidar for Meteorological Observations and The University of Western Ontario's Purple Crow Lidar.
Inhibition of G protein-activated inwardly rectifying K+ channels by extracts of Polygonum persicaria and isolation of new flavonoids from the chloroform extract of the herb.
Lajter, Ildik¾; Vasas, Andrea; Orvos, Pķter; Bßnsßghi, Szßva; Tßlosi, Lßszl¾; Jakab, Gusztßv; Bķni, Zoltßn; Hßda, Viktor; Forgo, Peter; Hohmann, Judit
The G protein-activated inwardly rectifying K+ channel-modulatory activities of Polygonum persicaria extracts were investigated by using an automated patch-clamp method, with the aim of identifying natural sources of promising ion channel-blocking compounds. The chloroform extract of the whole plant at 0.1 mg/mL exhibited high G protein-activated inwardly rectifying K+ channel-inhibitory activity. Fractionation of this extract by vacuum liquid chromatography on RP-silica gel resulted in 6 fractions, which were evaluated for G protein-activated inwardly rectifying K+ channel-modulatory activity. RP-HPLC of the most active fractions afforded the main compounds 1-4 in pure form and a mixture containing the minor constituents. The structures were identified by means of UV, HRMS, and advanced NMR methods as 3-O-senecioyl-isorhamnetin (1), 3-O-angeloyl-isorhamnetin (2), 5,3',4',5'-tetramethoxy-6,7-methylenedioxyflavone (3), and 3,5,3',4',5'-pentamethoxy-6,7-methylenedioxyflavone (4). Compounds 1-4 are new natural products, though 4 was reported earlier as a synthetic compound. Neither the individual, nor the combined application of compounds 1-4 modified the G protein-activated inwardly rectifying K+ channel activity. However, a marked G protein-activated inwardly rectifying K+ current-inhibitory effect was detected on use of the HPLC eluates containing the minor compounds. These results indicate the presence of electrophysiologically active agents among the minor compounds. PMID:24218371
Hydrophilic microenvironment required for the channel-independent insertase function of YidC protein.
Shimokawa-Chiba, Naomi; Kumazaki, Kaoru; Tsukazaki, Tomoya; Nureki, Osamu; Ito, Koreaki; Chiba, Shinobu
The recently solved crystal structure of YidC protein suggests that it mediates membrane protein insertion by means of an intramembrane cavity rather than a transmembrane (TM) pore. This concept of protein translocation prompted us to characterize the native, membrane-integrated state of YidC with respect to the hydropathic nature of its TM region. Here, we show that the cavity-forming region of the stage III sporulation protein J (SpoIIIJ), a YidC homolog, is indeed open to the aqueous milieu of the Bacillus subtilis cells and that the overall hydrophilicity of the cavity, along with the presence of an Arg residue on several alternative sites of the cavity surface, is functionally important. We propose that YidC functions as a proteinaceous amphiphile that interacts with newly synthesized membrane proteins and reduces energetic costs of their membrane traversal. PMID:25855636
A Communication interface using vesicles embedded with channel forming proteins in molecular communication
Y. Moritani; S. Hiyama; S. M. Nomura; K. Akiyoshi; T. Suda
This paper describes design and empirically study of a communication interface in molecular communication. The communication interface hides the characteristics of the molecules during the propagation from the sender to a receiver to allow a generic transport of molecules independent of the characteristics. The authors of this paper propose a communication interface that uses a vesicle embedded with channel forming
Construction, characterization, expression and immune responses of flagellar proteins of channel catfish, important pathogen Edwardsiella ictaluri
Technology Transfer Automated Retrieval System (TEKTRAN)
Background: Edwardsiella ictaluri causes enteric septicemia of catfish, which is the leading disease in channel catfish (Ictalurus punctatus)and is responsible for $50 - 60 million economic losses to catfish producers annually in the southeastern U.S. Bacterial flagella are complex polymeric structu...
Quantifying the effects of stream channels on storm water quality in a semi-arid urban environment
NASA Astrophysics Data System (ADS)
Gallo, Erika L.; Lohse, Kathleen A.; Brooks, Paul D.; McIntosh, Jennifer C.; Meixner, Thomas; McLain, Jean E. T.
SummaryStormwater drainage systems can have a large effect on urban runoff quality, but it is unclear how ephemeral urban streams alter runoff hydrochemistry. This problem is particularly relevant in semi-arid regions, where urban storm runoff is considered a renewable water resource. Here we address the question: how do stream channels alter urban runoff hydrochemistry? We collected synoptic stormwater samples during three rainfall-runoff events from nine ephemeral streams reaches (three concrete or metal, three grass, three gravel) in Tucson, Arizona. We identified patterns of temporal and spatial (longitudinal) variability in concentrations of conservative (chloride and isotopes of water) and reactive solutes (inorganic-N, soluble reactive phosphorous, sulfate-S, dissolved organic carbon (DOC) and nitrogen, and fecal indicator bacteria). Water isotopes and chloride (Cl) concentrations indicate that solute flushing and evapoconcentration alter temporal patterns in runoff hydrochemistry, but not spatial hydrochemical responses. Solute concentrations and stream channel solute sourcing and retention during runoff were significantly more variable at the grass reaches (CV = 2.3 - 144%) than at the concrete or metal (CV = 1.6 - 107%) or gravel reaches (CV = 1.9 - 60%), which functioned like flow-through systems. Stream channel soil Cl and DOC decreased following a runoff event (Cl: 12.1-7.3 ?g g-1 soil; DOC: 87.7-30.1 ?g g-1 soil), while soil fecal indicator bacteria counts increased (55-215 CFU g-1 soil). Finding from this study suggest that the characteristics of the ephemeral stream channel substrate control biogeochemical reactions between runoff events, which alter stream channel soil solute stores and the hydrochemistry of subsequent runoff events.
Effects of sugar, protein and water content on wheat starch gelatinization due to microwave heating
G. ?umnu; M. K. Ndife; L. Bay?nd?rl?
Understanding the interactions between sugar, starch, protein and water, which are the main components of a baked product,\\u000a will advance the development of high quality, microwaveable products. This paper presents a mathematical model describing\\u000a the quantitative relationships between water, sugar and protein on the gelatinization of wheat starch following 20?s of microwave\\u000a heat as determined by differential scanning calorimetry. Addition
Breakdown of the Debye polarization ansatz at protein-water interfaces.
Fernßndez Stigliano, Ariel
The topographical and physico-chemical complexity of protein-water interfaces scales down to the sub-nanoscale range. At this level of confinement, we demonstrate that the dielectric structure of interfacial water entails a breakdown of the Debye ansatz that postulates the alignment of polarization with the protein electrostatic field. The tendencies to promote anomalous polarization are determined for each residue type and a particular kind of structural defect is shown to provide the predominant causal context. PMID:23781824
The effects of water and heat stress on protein synthesis in loblolly pine (Pinus taeda L.)
Hulbert, Chrys
THE EFFECTS OF WATER AND HEAT STRESS ON PROTEIN SYNTHESIS IN LOBLOLLY PINE (PINUS TAEDA L. ) A Thesis CHRYS HULBERT Submitted to the Graduate College of Texas AAM University in partial fulfillment of the requirements for the degree... of MASTER OF SCIENCE May 1987 Major Subjett: Plant Physiology THE EFFECTS OF WATER AND HEAT STRESS ON PROTEIN SYNTHESIS IN LOBLOLLY PINE (PINUS TAEDA L. ) A Thesis CHRYS HULBERT Approved as to style and content by: Ronald . Newton I (Co...
Effect of protein and glycerol concentration on the mechanical, optical, and water vapor barrier properties of canola protein isolate-based edible films.
Chang, Chang; Nickerson, Michael T
Biodegradable edible films prepared using proteins are both economically and environmentally important to the food packaging industry relative to traditional petroleum-derived synthetic materials. In the present study, the mechanical and water vapor barrier properties of casted canola protein isolate edible films were investigated as a function of protein (5.0% and 7.5%) and glycerol (30%, 35%, 40%, 45%, and 50%) content. Specifically, tensile strength and elongation, elastic modulus, puncture strength and deformation, opacity, and water vapor permeability were measured. Results indicated that tensile strength, puncture strength, and elastic modulus decreased, while tensile elongation and puncture deformation values increased as glycerol concentration increased for both 5.0% and 7.5% canola protein isolate films. Furthermore, tensile strength, puncture strength, and elastic modulus values were found to increase at higher protein concentrations within the canola protein isolate films, whereas puncture deformation values decreased. Tensile elongation was found to be similar for both canola protein isolate protein levels. Canola protein isolate films became more transparent with increasing of glycerol concentration and decreasing of canola protein isolate concentration. Water vapor permeability value was also found to increase with increasing glycerol and protein contents. Overall, results indicated that canola protein isolate films were less brittle, more malleable and transparent, and had greater water vapor permeability at higher glycerol levels. However, as protein level increased, canola protein isolate films were more brittle, less malleable and more opaque, and also had increased water vapor permeability. PMID:24072788
Interlayer Water Regulates the Bio-nano Interface of a \\b{eta}-sheet Protein stacking on Graphene
Wenping Lv; Guiju Xu; Hongyan Zhang; Xin Li; Shengju Liu; Huan Niu; Dongsheng Xu; Renan Wu
Using molecular dynamics simulations, we investigated an integrated bio-nano interface consisting of a \\b{eta}-sheet protein stacked onto graphene. We found that the stacking assembly of the model protein on graphene could be controlled by water molecules. The interlayer water filled within interstices of the bio-nano interface could suppress the molecular vibration of surface groups on protein, and could impair the CH...{\\pi} interaction driving the attraction of the protein and graphene. The intermolecular coupling of interlayer water would be relaxed by the relative motion of protein upon graphene due to the interaction between water and protein surface. This effect reduced the hindrance of the interlayer water against the assembly of protein on graphene, resulting an appropriate adsorption status of protein on graphene with a deep free energy trap. Thereby, the confinement and the relative sliding between protein and graphene, the coupling of protein and water, and the interaction between graphene and water all have involved in the modulation of behaviors of water molecules within the bio-nano interface, governing the hindrance of interlayer water against the protein assembly on hydrophobic graphene. These results provide a deep insight into the fundamental mechanism of protein adsorption onto graphene surface in water.
Molecular Dynamics Analysis of Lysozyme Protein in Ethanol-Water Mixed Solvent Environment
NASA Astrophysics Data System (ADS)
Ochije, Henry Ikechukwu
Effect of protein-solvent interaction on the protein structure is widely studied using both experimental and computational techniques. Despite such extensive studies molecular level understanding of proteins and some simple solvents is still not fully understood. This work focuses on detailed molecular dynamics simulations to study of solvent effect on lysozyme protein, using water, alcohol and different concentrations of water-alcohol mixtures as solvents. The lysozyme protein structure in water, alcohol and alcohol-water mixture (0-12% alcohol) was studied using GROMACS molecular dynamics simulation code. Compared to water environment, the lysozome structure showed remarkable changes in solvents with increasing alcohol concentration. In particular, significant changes were observed in the protein secondary structure involving alpha helices. The influence of alcohol on the lysozyme protein was investigated by studying thermodynamic and structural properties. With increasing ethanol concentration we observed a systematic increase in total energy, enthalpy, root mean square deviation (RMSD), and radius of gyration. a polynomial interpolation approach. Using the resulting polynomial equation, we could determine above quantities for any intermediate alcohol percentage. In order to validate this approach, we selected an intermediate ethanol percentage and carried out full MD simulation. The results from MD simulation were in reasonably good agreement with that obtained using polynomial approach. Hence, the polynomial approach based method proposed here eliminates the need for computationally intensive full MD analysis for the concentrations within the range (0-12%) studied in this work.
Water-holding capacity and protein denatunation in broiler breast meat
Technology Transfer Automated Retrieval System (TEKTRAN)
The aim of this study was to investigate the impact of protein denaturation on water-holding capacity (WHC) in broiler breast meat. Breast fillets were collected at 2 h postmortem and segregated into two groups (low-WHC and high-WHC) based on pH and color. Protein solubility was measured at 6 and 24...
Transcapillary Water and Protein Flux in the Canine Intestine with Acute and Chronic Extrahepatic Portal Hypertension
Charles L. Witte; John F. Myers; Marlys H. Witte; Murray A. Katz
SUMMARY. Intestinal transcapillary water and total protein flux were determined in dogs with chronic extrahepatic portal hypertension after construction of an aortic-portal shunt combined with hilar portal vein constriction and compared to acute portal vein constriction. Measurements were made of thoracic duct lymph flow, portal venous pressure, and total protein concentration in plasma, thoracic duct lymph, intestinal and liver lymph.
Extraction of protein and amino acids from deoiled rice bran by subcritical water hydrolysis
Issara Sereewatthanawut; Surawit Prapintip; Katemanee Watchiraruji; Motonobu Goto; Mitsuru Sasaki; Artiwan Shotipruk
This study investigated the production of value-added protein and amino acids from deoiled rice bran by hydrolysis in subcritical water (SW) in the temperature range between 100 and 220░C for 0¢30min. The results suggested that SW could effectively be used to hydrolyze deoiled rice bran to produce useful protein and amino acids. The amount of protein and amino acids produced
Water vapor permeability properties of edible whey protein-lipid emulsion films
Tara Habig McHugh; John M. Krochta
The water vapor permeability (WVP) of whey protein emulsion films was investigated. The exponential effect of relative humidity\\u000a on the WVP of whey protein films was reduced through lipid incorporation. Film orientation had a significant effect on WVP\\u000a due to emulsion separation during film formation. Heat denaturation of whey proteins lowered emulsion film WVP. Increasing\\u000a fatty acid and fatty alcohol
Cell-Wall Proteins Induced by Water Deficit in Bean (Phaseolus vulgaris L.) Seedlings.
Covarrubias, A. A.; Ayala, J. W.; Reyes, J. L.; Hernandez, M.; Garciarrubio, A.
In the last few years, much attention has been given to the role of proteins that accumulate during water deficit. In this work, we analyzed the electrophoretic patterns of basic protein extracts, enriched for a number of cell-wall proteins, from bean (Phaseolus vulgaris L.) seedlings and 21-d-old plants subjected to water deficit. Three major basic proteins accumulated in bean seedlings exposed to low water potentials, with apparent molecular masses of 36, 33, and 22 kD, which we refer to as p36, p33, and p22, respectively. Leaves and roots of 21-d-old plants grown under low-water-availability conditions accumulated only p36 and p33 proteins. In 21-d-old plants subjected to a fast rate of water loss, both p33 and p36 accumulated to approximately the same levels, whereas if the plants were subjected to a gradual loss of water, p33 accumulated to higher levels. Both p36 and p33 were glycosylated and were found in the cell-wall fraction. In contrast, p22 was not glycosylated and was found in the soluble fraction. The accumulation of these proteins was also induced by abscisic acid (0.1-1.0 mM) treatment but not by wounding or by jasmonate treatment. PMID:12228420
Copper-based micro-channel cooler reliably operated using solutions of distilled-water and ethanol as a coolant
NASA Astrophysics Data System (ADS)
Chin, A. K.; Nelson, A.; Chin, R. H.; Bertaska, R.; Jacob, J. H.
Copper-based micro-channel coolers (Cu-MCC) are the lowest thermal-resistance heat-sinks for high-power laserdiode (LD) bars. Presently, the resistivity, pH and oxygen content of the de-ionized water coolant, must be actively controlled to minimize cooler failure by corrosion and electro-corrosion. Additionally, the water must be constantly exposed to ultraviolet radiation to limit the growth of micro-organisms that may clog the micro-channels. In this study, we report the reliable, care-free operation of LD-bars attached to Cu-MCCs, using a solution of distilledwater and ethanol as the coolant. This coolant meets the storage requirements of Mil-Std 810G, e.g. exposure to a storage temperature as low as -51░C and no growth of micro-organisms during passive storage.
G-protein-coupled receptors, channels, and Na+-H+ exchanger in nuclear membranes of heart, hepatic, vascular endothelial, and smooth muscle cells.
Bkaily, Ghassan; Nader, Moni; Avedanian, Levon; Choufani, Sana; Jacques, Danielle; D'Orlķans-Juste, Pedro; Gobeil, Fernand; Chemtob, Sylvain; Al-Khoury, Johny
The action of several peptides and drugs is thought to be primarily dependent on their interactions with specific cell surface G-protein-coupled receptors and ionic transporters such as channels and exchangers. Recent development of 3-D confocal microscopy allowed several laboratories, including ours, to identify and study the localization of receptors, channels, and exchangers at the transcellular level of several cell types. Using this technique, we demonstrated in the nuclei of several types of cells the presence of Ca(2+) channels as well as Na(+)-H(+) exchanger and receptors such as endothelin-1 and angiotensin II receptors. Stimulation of these nuclear membrane G-protein-coupled receptors induced an increase of nuclear Ca(2+). Our results suggest that, similar to the plasma membrane, nuclear membranes possess channels, exchangers and receptors such as those for endothelin-1 and angiotensin II, and that the nucleus seems to be a cell within a cell. This article will emphasize these findings. PMID:16902588
Activation of kappa-opioid receptor as a method for prevention of ischemic and reperfusion arrhythmias: role of protein kinase C and K(ATP) channels.
Lishmanov, A Yu; Maslov, L N; Lasukova, T V; Crawford, D; Wong, T M
Intravenous pretreatment with kappa-opioid receptor antagonist (-)-U-50,488 (1 mg/kg) improved heart resistance to the arrhythmogenic effect of coronary occlusion and reperfusion. Selective kappa1-opioid receptor antagonist norbinaltorphimine and nonselective blocker of peripheral opioid receptors methylnaloxone abolished this antiarrhythmic effect. Preliminary blockade of protein kinase C with chelerythrine or inhibition of ATP-dependent K+ channels (K(ATP) channels) with glybenclamide abolished the antiarrhythmic effect of kappa-opioid receptor activation. Selective inhibitor of sarcolemmal K(ATP) channels did not modulate the kappa-opioid receptor-mediated increase in cardiac electrical stability. Our results suggest that protein kinase C and mitochondrial K(ATP) channels play an important role in the antiarrhythmic effect associated with activation of peripheral kappa-opioid receptors. PMID:17970197
Brain expression of the water channels Aquaporin1 and -4 in mice with acute liver injury, hyperammonemia and brain edema
Martin Eefsen; Peter Jelnes; Lars E. Schmidt; Ben Vainer; Hanne Cathrine Bisgaard; Fin S. Larsen
Cerebral edema is a feared complication to acute liver failure (ALF), but the pathogenesis is still poorly understood. The\\u000a water channels Aquaporin-1 (Aqp1) and -4 (Aqp4) has been associated with brain edema formation in several neuropathological\\u000a conditions, indicating a possible role of Aqp1 and\\/or Aqp4 in ALF mediated brain edema. We induced acute liver injury and\\u000a hyperammonemia in mice, to
The role of intercellular channels in the transepithelial transfer of water and sodium in the frog urinary bladder
S. Jard; J. Bourguet; p. Favard; N. Carasso
SummaryĀĀEpithelial cells of frog urinary bladders fixed in different physiological states were examined by electron microscopy. It\\u000a was shown: (1) that when bladders incubated with a hypotonic mucosal medium are water-permeabilized with oxytoxin, arginine-vasotocin,\\u000a cyclic 3?,5?-AMP and theophylline, this leads to a cellular swelling and the opening of intercellular channels; (2) that these\\u000a effects are not observed when the transepithelial
Design and evaluation of hydraulic baffled-channel PAC contactor for taste and odor removal from drinking water supplies.
Kim, Young-Il; Bae, Byung-Uk
Based on the concept of hydraulic flocculator, a baffled-channel powdered activated carbon (PAC) contactor, placed before the rapid-mixing basin, was designed and evaluated for removal of taste and odor (T&O) in drinking water. PAC adsorption kinetic tests for raw water samples were conducted for selection of design parameters related to contact time and degree of mixing. Within the tested range of velocity gradient (G) from 18 to 83s(-1), mixing had a relatively minor effect on the adsorption kinetics of the PAC. The hydrodynamic characteristics of the pilot-scale horizontally and vertically baffled-channel PAC contactor were investigated by tracer tests. It was found that the plug flow fractions of vertically baffled-channel PAC contactor (vBPC) were higher than those of the horizontally baffled-channel PAC contactor (hBPC) for the same bend width or bend height. However, the hBPC seems to be more appropriate than the vBPC in terms of construction and maintenance. The geosmin and MIB removal rate increased with the number of baffles, PAC dose and contact time increased regardless of bend width in the pilot-scale hBPC. The pair of full-scale hBPCs at Pohang water treatment plant, having a design capacity of 6.5x10(4)m(3)/d with 20min of hydraulic retention time with a safety factor of 2, was designed based on lab- and pilot-scale experimental results. Under a velocity gradient of 20s(-1), the number of baffles to be installed was calculated to be 20 with a space of about 2m between each baffle, resulting in a hydraulic head loss through the contactor of about 0.056m. The successful application of hBPC for T&O removal from drinking water supplies should provide momentum for developing more effective treatment methods. PMID:17400274
In silico studies of the properties of water hydrating a small protein.
Sinha, Sudipta Kumar; Jana, Madhurima; Chakraborty, Kausik; Bandyopadhyay, Sanjoy
Atomistic molecular dynamics simulation of an aqueous solution of the small protein HP-36 has been carried out with explicit solvent at room temperature. Efforts have been made to explore the influence of the protein on the relative packing and ordering of water molecules around its secondary structures, namely, three ?-helices. The calculations reveal that the inhomogeneous water ordering and density distributions around the helices are correlated with their relative hydrophobicity. Importantly, we have identified the existence of a narrow relatively dehydrated region containing randomly organized "quasi-free" water molecules beyond the first layer of "bound" waters at the protein surface. These water molecules with relatively weaker binding energies form the transition state separating the "bound" and "free" water molecules at the interface. Further, increased contribution of solid-like caging motions of water molecules around the protein is found to be responsible for reduced fluidity of the hydration layer. Interestingly, we notice that the hydration layer of helix-3 is more fluidic with relatively higher entropy as compared to the hydration layers of the other two helical segments. Such characteristics of helix-3 hydration layer correlate well with the activity of HP-36, as helix-3 contains the active site of the protein. PMID:25494773
In silico studies of the properties of water hydrating a small protein
Sinha, Sudipta Kumar; Chakraborty, Kausik; Bandyopadhyay, Sanjoy, E-mail: email@example.com [Molecular Modeling Laboratory, Department of Chemistry, Indian Institute of Technology, Kharagpur - 721302 (India); Jana, Madhurima [Molecular Simulation Laboratory, Department of Chemistry, National Institute of Technology, Rourkela - 769008 (India)
Atomistic molecular dynamics simulation of an aqueous solution of the small protein HP-36 has been carried out with explicit solvent at room temperature. Efforts have been made to explore the influence of the protein on the relative packing and ordering of water molecules around its secondary structures, namely, three ?-helices. The calculations reveal that the inhomogeneous water ordering and density distributions around the helices are correlated with their relative hydrophobicity. Importantly, we have identified the existence of a narrow relatively dehydrated region containing randomly organized ōquasi-freeö water molecules beyond the first layer of ōboundö waters at the protein surface. These water molecules with relatively weaker binding energies form the transition state separating the ōboundö and ōfreeö water molecules at the interface. Further, increased contribution of solid-like caging motions of water molecules around the protein is found to be responsible for reduced fluidity of the hydration layer. Interestingly, we notice that the hydration layer of helix-3 is more fluidic with relatively higher entropy as compared to the hydration layers of the other two helical segments. Such characteristics of helix-3 hydration layer correlate well with the activity of HP-36, as helix-3 contains the active site of the protein.
In silico studies of the properties of water hydrating a small protein
NASA Astrophysics Data System (ADS)
Sinha, Sudipta Kumar; Jana, Madhurima; Chakraborty, Kausik; Bandyopadhyay, Sanjoy
Atomistic molecular dynamics simulation of an aqueous solution of the small protein HP-36 has been carried out with explicit solvent at room temperature. Efforts have been made to explore the influence of the protein on the relative packing and ordering of water molecules around its secondary structures, namely, three ?-helices. The calculations reveal that the inhomogeneous water ordering and density distributions around the helices are correlated with their relative hydrophobicity. Importantly, we have identified the existence of a narrow relatively dehydrated region containing randomly organized "quasi-free" water molecules beyond the first layer of "bound" waters at the protein surface. These water molecules with relatively weaker binding energies form the transition state separating the "bound" and "free" water molecules at the interface. Further, increased contribution of solid-like caging motions of water molecules around the protein is found to be responsible for reduced fluidity of the hydration layer. Interestingly, we notice that the hydration layer of helix-3 is more fluidic with relatively higher entropy as compared to the hydration layers of the other two helical segments. Such characteristics of helix-3 hydration layer correlate well with the activity of HP-36, as helix-3 contains the active site of the protein.
Spinocerebellar ataxia-13 Kv3.3 potassium channels: arginine-to-histidine mutations affect both functional and protein expression on the cell surface.
Zhao, Jian; Zhu, Jing; Thornhill, William B
The voltage-gated potassium channel Kv3.3 is the causative gene of SCA13 (spinocerebellar ataxia type 13), an autosomal dominant neurological disorder. The four dominant mutations identified to date cause Kv3.3 channels to be non-functional or have altered gating properties in Xenopus oocytes. In the present paper, we report that SCA13 mutations affect functional as well as protein expression of Kv3.3 channels in a mammalian cell line. The reduced protein level of SCA13 mutants is caused by a shorter protein half-life, and blocking the ubiquitin-proteasome pathway increases the total protein of SCA13 mutants more than wild-type. SCA13 mutated amino acids are highly conserved, and the side chains of these residues play a critical role in the stable expression of Kv3.3 proteins. In addition, we show that mutant Kv3.3 protein levels could be partially rescued by treatment with the chemical chaperone TMAO (trimethylamine N-oxide) and to a lesser extent with co-expression of Kv3.1b. Thus our results suggest that amino acid side chains of SCA13 positions affect the protein half-life and/or function of Kv3.3, and the adverse effect on protein expression cannot be fully rescued. PMID:23734863
H{sub 2}O monomers in channels of icelike water structures
Pershin, S. M., E-mail: email@example.com; Bunkin, A. F. [Russian Academy of Sciences, Prokhorov General Physics Institute (Russian Federation); Golo, V. L., E-mail: firstname.lastname@example.com [Moscow State University (Russian Federation)
We consider the dynamics of H{sub 2}O monomers in channels of icelike structures using the model of 1D rotator (i.e., a particle with one translational and one rotational degree of freedom). The effect of the channel walls on the motion of the particle is simulated by the interaction of the dipole moment of the particle with a periodic electric field. In this model, the following four regimes of motion are possible: (i) reflection of the particle from the inlet of the channel; (ii) single passage through the channel with a fixed phase; (iii) passage of the particle through the channel after multiple reflections in the channel from its inlet to its outlet; and (iv) reverse motion of the particle after its stay in the channel and multiple reflections from the inlet and outlet. These regimes indicate the possibility of existence of H{sub 2}O monomers in the form of particles trapped in the channels of icelike formations.
Blade design and performance analysis on the horizontal axis tidal current turbine for low water level channel
NASA Astrophysics Data System (ADS)
Chen, C. C.; Choi, Y. D.; Y Yoon, H.
Most tidal current turbine design are focused on middle and large scale for deep sea, less attention was paid in low water level channel, such as the region around the islands, coastal seas and rivers. This study aims to develop a horizontal axis tidal current turbine rotor blade which is applicable to low water level island region in southwest of Korea. The blade design is made by using BEMT(blade element momentum theory). The section airfoil profile of NACA63-415 is used, which shows good performance of lift coefficient and drag coefficient. Power coefficient, pressure and velocity distributions are investigated according to TSR by CFD analysis.
Influenza A Virus M2 Protein: Proton Selectivity of the Ion Channel, Cytotoxicity, and a Hypothesis on Peripheral Raft Association and Virus Budding
Cornelia Schroeder; Tse-I Lin
The influenza A virus M2 protein, the prototype viral ion channel, mediates passage through low-pH compartments during viral\\u000a entry and maturation. Its proton channel activity is essential for virus uncoating and in certain cases for the maturation\\u000a of viral hemagglutinin (HA). A fluorimetric assay of ion translocation by membrane-reconstituted M2 disclosed the nature of\\u000a the conducted ions, protons, and allowed
Defective dietary fat processing in transgenic mice lacking aquaporin-1 water channels
Immunocytochemistry showed expression of aquaporin-1 (AQP1) water channels at sites involved in dietary fat processing, including intrahepatic cholangiocytes, gallbladder, pancreatic microvascular endothelium, and intestinal lacteals. To determine whether AQP1 has a role in dietary fat digestion and/or absorption, mice were placed on a diet that contained 50% fat. Whereas wild-type mice (3¢3.5 wk of age, 10¢12 g) gained 49 ▒ 5% (SE, n = 50) body weight in 8 days, and heterozygous mice gained 46 ▒ 4%, AQP1 null mice gained only 4 ▒ 3%; weights became similar after return to a 6% fat diet after 6 days. The null mice on a high-fat diet acquired an oily appearance, developed steatorrhea with increased stool triglyceride content, and manifested serum hypotriglyceri-demia. Supplementation of the high-fat diet with pancreatic enzymes partially corrected the decreased weight gain in null mice. Absorption of [14C]oleic acid from small intestine was not affected by AQP1 deletion, as determined by blood radio-activity after duodenal infusion. Lipase activity in feces and small intestine was remarkably greater in AQP1 null than wild-type mice on low- and high-fat diets. Fluid collections done in older mice (that are less sensitive to a high-fat diet) by ductal cannulation showed threefold increased pancreatic fluid flow in response to secretin/cholecystokinin, but volumes, pH, and amylase activities were affected little by AQP1 deletion, nor were bile flow rates and bile salt concentrations. Together, these results establish a dietary fat misprocessing defect in AQP1 null mice. PMID:11121384
Analysis of ocean color components within stratified and well-mixed waters of the western English Channel
NASA Technical Reports Server (NTRS)
Hochmann, Herschel T.; Walsh, John J.; Carder, Kendall L.; Sournia, A.; Muller-Karger, Frank E.
In situ pigment and dissolved organic carbon (DOC) data from two distinct hydrographic regions of the western English Channel are used to explore the possible marine DOC contamination of the past satellite estimates of phytoplankton biomass. To compare with field measurements, the individual spectral contributions of DOC, pigments, and water to the total diffuse attenuation coefficient, K(sub par), are summed on a quantum basis within stratified waters near Plymouth, England; and for the spectrally averaged diffuse attenuation coefficient, K(sub d), on an energy basis within tidally mixed waters near Roscoff, France. In addition, coastal zone color scanner (CZCS) images from 1979 to 1986 were used to compute DOC concentrations for comparison with in situ values. Our analysis suggests that almost 50% of the color signal of satellite-sensed pigments may be attributed to absorption by marine colored DOC (CDOC) within the English Channel. These results compare favorably to the in situ DOC measurements off Plymouth, but not to off-Roscoff measurements, suggesting that there may be more CDOC in the stratified waters and more nonabsorbing DOC in the tidally mixed waters.
Quality of water in the Black River near Dunn, North Carolina, and ground-water levels adjacent to the river prior to channel excavation in 1978-79
Simmons, Clyde E.
During 1976-79 data were collected at three sites on the Black River, near Dunn, North Carolina, to define water-quality and other hydrologic conditions prior to channel excavation. Samples collected over a range in flow from 1.2 to 900 cubic feet per second contained 1 to 81 mg/L (milligrams per liter) of suspended sediment, 37 to 108 mg/L of dissolved solids, and 0.21 to 1.0 mg/L of total nitrogen. Water-level fluctuations in wells located within 100 feet of the Black River were almost identical with those of the stream.
Identification of Characteristic Protein Folding Channels in a Coarse-Grained Hydrophobic-Polar Peptide Model
Stefan Schnabel; Michael Bachmann; Wolfhard Janke
Folding channels and free-energy landscapes of hydrophobic-polar heteropolymers are discussed on the basis of a minimalistic off-lattice coarse-grained model. We investigate how rearrangements of hydrophobic and polar monomers in a heteropolymer sequence lead to completely different folding behaviors. Studying three exemplified sequences with the same content of hydrophobic and polar residues, we can reproduce within this simple model two-state folding, folding through intermediates, as well as metastability.
MBNL and CELF proteins regulate alternative splicing of the skeletal muscle chloride channel CLCN1
Yoshihiro Kino; Chika Washizu; Yoko Oma; Hayato Onishi; Yuriko Nezu; Noboru Sasagawa; Nobuyuki Nukina; Shoichi Ishiura
The expression and function of the skeletal muscle chloride channel CLCN1\\/ClC-1 is regulated by alter- native splicing. Inclusion of the CLCN1 exon 7A is aberrantly elevated in myotonic dystrophy (DM), a genetic disorder caused by the expansion of a CTG or CCTG repeat. Increased exon 7A inclusion leads to a reduction in CLCN1 function, which can be causative of myotonia.
Identification of compounds with binding affinity to proteins via magnetization transfer from bulk water
Claudio Dalvit; Paolo Pevarello; Marco Tat"; Marina Veronesi; Anna Vulpetti; Michael Sundstr÷m
A powerful screening by NMR methodology (WaterLOGSY), based on transfer of magnetization from bulk water, for the identification of compounds that interact with target biomolecules (proteins, RNA and DNA fragments) is described. The method exploits efficiently the large reservoir of H2O magnetization. The high sensitivity of the technique reduces the amount of biomolecule and ligands needed for the screening, which
Water quality model parameter identification of an open channel in a long distance water transfer project based on finite difference, difference evolution and Monte Carlo.
Shao, Dongguo; Yang, Haidong; Xiao, Yi; Liu, Biyu
A new method is proposed based on the finite difference method (FDM), differential evolution algorithm and Markov Chain Monte Carlo (MCMC) simulation to identify water quality model parameters of an open channel in a long distance water transfer project. Firstly, this parameter identification problem is considered as a Bayesian estimation problem and the forward numerical model is solved by FDM, and the posterior probability density function of the parameters is deduced. Then these parameters are estimated using a sampling method with differential evolution algorithm and MCMC simulation. Finally this proposed method is compared with FDM-MCMC by a twin experiment. The results show that the proposed method can be used to identify water quality model parameters of an open channel in a long distance water transfer project under different scenarios better with fewer iterations, higher reliability and anti-noise capability compared with FDM-MCMC. Therefore, it provides a new idea and method to solve the traceability problem in sudden water pollution accidents. PMID:24552732
Interaction of Globular Plasma Proteins with Water-Soluble CdSe Quantum Dots.
Pathak, Jyotsana; Rawat, Kamla; Sanwlani, Shilpa; Bohidar, H B
The interactions between water-soluble semiconductor quantum dots [hydrophilic 3-mercaptopropionic acid (MPA)-coated CdSe] and three globular plasma proteins, namely, bovine serum albumin (BSA), ?-lactoglobulin (?-Lg) and human serum albumin (HSA), are investigated. Acidic residues of protein molecules form electrostatic interactions with these quantum dots (QDs). To determine the stoichiometry of proteins bound to QDs, we used dynamic light scattering (DLS) and zeta potential techniques. Fluorescence resonance energy transfer (FRET) experiments revealed energy transfer from tryptophan residues in the proteins to the QD particles. Quenching of the intrinsic fluorescence of protein molecules was noticed during this binding process (hierarchy HSA-Lg
Moisture-induced quality changes of hen egg white proteins in a protein/water model system.
Rao, Qinchun; Rocca-Smith, Jeancarlo R; Labuza, Theodore P
In recent years, the intermediate-moisture foods (IMF), such as nutrition and energy bars, are a rapidly growing segment of the global food market. However, due to moisture-induced protein aggregation, commercial high protein nutrition bars generally become harder over time, thus losing product acceptability. In this study, the objectives were to investigate the moisture-induced protein aggregation in a hen egg white proteins/water dough model system (water activity (a(w)): 0.95) and to evaluate its molecular mechanisms and controlling factors. During storage at three different temperatures (23, 35, and 45 ░C) for 70 days, four selected physicochemical changes of the dough system were analyzed: the a(w), the color (L* value), the fluorescent Maillard compounds (fluorescence intensity (FI) value), and the remaining free amino groups. Overall, the physicochemical changes of egg white proteins in the dough system are closely related to the glass transition temperature (T(g)). The effect of moisture content on both the L* and FI values occurred as a function of storage time at 45 ░C due to the Maillard reaction. The change of the remaining free amino groups at different temperatures was derived from the coaction of both the Maillard reaction and enzymatic hydrolysis from molds. Additionally, through analyzing the buffer-soluble egg white proteins using gel electrophoresis, our results showed that moisture-induced aggregates were produced by two chemical reactions during storage: the disulfide interaction and the Maillard reaction. Furthermore, the effect of two processes during manufacturing, desugarization and dry-heat pasteurization, on the physicochemical changes of the egg white proteins was elucidated. In order to prevent or reduce moisture-induced protein aggregation during product storage and distribution, two potential solutions were also discussed. PMID:22985260
Effect of water salinity on total protein and electrophoretic pattern of serum proteins of grass carp, Ctenopharyngodon idella.
Peyghan, Rahim; Khadjeh, Gholam Hosain; Enayati, Ala
In this study the effects of water salinity on serum total protein and its components in grass carp were investigated. The aim of this study was to determine the effect of salinity tolerance of fish on total serum protein level and its components as an indicator of liver and kidney activity. One hundred and twenty grass carp were divided into four groups, randomly. The first three groups were reared in concentration of 4, 8 and 12 g L(-1) of salt solution, respectively, and the fourth group was reared in freshwater and served as control. After 3 weeks, blood samples were collected and after harvesting the blood serum, serum total protein and protein components were measured with Biuret and electrophoresis methods, respectively. Results showed that mean value of serum total protein in the control and three salinities groups were 2.75, 3.28, 2.90 and 3.13 g dL(-1), respectively. Five fractions of serum protein were electrophoretically observed as: albumin (Alb), alpha-1 globulin (?1-glu), alpha-2 globulin (?2-glu), beta globulin (?-glu) and gamma globulin (?-glu). There were not any significant differences between the average mean of serum total protein of experimental and control groups (p > 0.05). However, Alb, ?1-glu and ?-glu levels in the experimental groups were significantly higher than in the control group (p < 0.05). The average of ?2-glu and ?-glu revealed no significant difference between the experimental groups (p > 0.05). In conclusion, our results showed that increasing water salinity could have a significant effect on Alb, ?1-glu and ?-glu levels but not on total serum protein in grass carp. PMID:25568723
SLITHER: a web server for generating contiguous conformations of substrate molecules entering into deep active sites of proteins or migrating through channels in membrane transporters
Lee, Po-Hsien; Kuo, Kuei-Ling; Chu, Pei-Ying; Liu, Eric M.; Lin, Jung-Hsin
Many proteins use a long channel to guide the substrate or ligand molecules into the well-defined active sites for catalytic reactions or for switching molecular states. In addition, substrates of membrane transporters can migrate to another side of cellular compartment by means of certain selective mechanisms. SLITHER (http://bioinfo.mc.ntu.edu.tw/slither/or http://slither.rcas.sinica.edu.tw/) is a web server that can generate contiguous conformations of a molecule along a curved tunnel inside a protein, and the binding free energy profile along the predicted channel pathway. SLITHER adopts an iterative docking scheme, which combines with a puddle-skimming procedure, i.e. repeatedly elevating the potential energies of the identified global minima, thereby determines the contiguous binding modes of substrates inside the protein. In contrast to some programs that are widely used to determine the geometric dimensions in the ion channels, SLITHER can be applied to predict whether a substrate molecule can crawl through an inner channel or a half-channel of proteins across surmountable energy barriers. Besides, SLITHER also provides the list of the pore-facing residues, which can be directly compared with many genetic diseases. Finally, the adjacent binding poses determined by SLITHER can also be used for fragment-based drug design. PMID:19433508
Crystal Structure of the Mammalian GIRK2 K[superscript +] Channel and Gating Regulation by G Proteins, PIP[subscript 2], and Sodium
Whorton, Matthew R.; MacKinnon, Roderick (Rockefeller)
G protein-gated K{sup +} channels (Kir3.1-Kir3.4) control electrical excitability in many different cells. Among their functions relevant to human physiology and disease, they regulate the heart rate and govern a wide range of neuronal activities. Here, we present the first crystal structures of a G protein-gated K{sup +} channel. By comparing the wild-type structure to that of a constitutively active mutant, we identify a global conformational change through which G proteins could open a G loop gate in the cytoplasmic domain. The structures of both channels in the absence and presence of PIP{sub 2} suggest that G proteins open only the G loop gate in the absence of PIP{sub 2}, but in the presence of PIP{sub 2} the G loop gate and a second inner helix gate become coupled, so that both gates open. We also identify a strategically located Na{sup +} ion-binding site, which would allow intracellular Na{sup +} to modulate GIRK channel activity. These data provide a structural basis for understanding multiligand regulation of GIRK channel gating.
Vibrational excitations of proteins and their hydration water in the far-infrared range
NASA Astrophysics Data System (ADS)
Paciaroni, A.; Conti Nibali, V.; Orecchini, A.; Petrillo, C.; Haertlein, M.; Moulin, M.; Tarek, M.; D'Angelo, G.; Sacchetti, F.
Incoherent neutron scattering has been used to single out the vibrational contribution from maltose binding protein (MBP) and its hydration water in the energy range 1 meV-80 meV. The vibrational density of states from both protein and hydration water have been investigated by measuring respectively dry and D2O-hydrated isotopically natural MBP and dry and H2O-hydrated perdeuterated MBP. Molecular dynamics simulations done on the same system allow us to attribute the protein inelastic features. The inelastic behavior of the biomolecule seems to be largely independent on the presence of solvent. Conversely, protein hydration water exhibits remarkable differences with respect to hexagonal ice in the whole spectral range, with clear similarities to amorphous phases of ice.
A two-channel, tunable diode laser-based hygrometer for measurement of water vapor and cirrus cloud ice water content in the upper troposphere and lower stratosphere
NASA Astrophysics Data System (ADS)
Thornberry, T. D.; Rollins, A. W.; Gao, R. S.; Watts, L. A.; Ciciora, S. J.; McLaughlin, R. J.; Fahey, D. W.
The recently developed NOAA Water instrument is a two-channel, closed-path, tunable diode laser absorption spectrometer designed for the measurement of water vapor and enhanced total water (vapor + inertially enhanced condensed-phase) in the upper troposphere/lower stratosphere from the NASA Global Hawk unmanned aircraft system (UAS) or other high-altitude research aircraft. The instrument utilizes wavelength-modulated spectroscopy with second harmonic detection near 2694 nm to achieve high precision with a 79 cm double-pass optical path. The detection cells are operated under constant temperature, pressure and flow conditions to maintain a constant sensitivity to H2O independent of the ambient sampling environment. An on-board calibration system is used to perform periodic in situ calibrations to verify the stability of the instrument sensitivity during flight. For the water vapor channel, ambient air is sampled perpendicular to the flow past the aircraft in order to reject cloud particles, while the total water channel uses a heated, forward-facing inlet to sample both water vapor and cloud particles. The total water inlet operates subisokinetically, thereby inertially enhancing cloud particle number in the sample flow and affording increased cloud water content sensitivity. The NOAA Water instrument was flown for the first time during the second deployment of the Airborne Tropical TRopopause EXperiment (ATTREX) in February-March 2013 on board the Global Hawk UAS. The instrument demonstrated a typical in-flight precision (1 s, 1?) of better than 0.17 parts per million (ppm, 10-6 mol mol-1), with an overall H2O vapor measurement uncertainty of 5% ▒ 0.23 ppm. The inertial enhancement for cirrus cloud particle sampling under ATTREX flight conditions ranged from 33-48 for ice particles larger than 8 ?m in diameter, depending primarily on aircraft altitude. The resulting ice water content detection limit (2?) was 0.023-0.013 ppm, corresponding to approximately 2 ?g m-3, with an estimated overall uncertainty of 20%.
Technology Transfer Automated Retrieval System (TEKTRAN)
This book chapter provides a comprehensive overview of channel catfish aquaculture. Sections include fish biology; commercial culture; culture facilities; production practices; water quality management; nutrition, feeding and feed formulation; infectious diseases; harvesting and processing; and the...
Activation of K+ channels in renal medullary vesicles by cAMP-dependent protein kinase
Reeves, W.B.; McDonald, G.A.; Mehta, P.; Andreoli, T.E. (Univ. of Arkansas College of Medicine, Little Rock (USA))
ADH, acting through cAMP, increases the potassium conductance of apical membranes of mouse medullary thick ascending limbs of Henle. The present studies tested whether exposure of renal medullary apical membranes in vitro to the catalytic subunit of cAMP-dependent protein kinase resulted in an increase in potassium conductance. Apical membrane vesicles prepared from rabbit outer renal medulla demonstrated bumetanide- and chloride-sensitive {sup 22}Na+ uptake and barium-sensitive, voltage-dependent {sup 86}Rb+ influx. When vesicles were loaded with purified catalytic subunit of cAMP-dependent protein kinase (150 mU/ml), 1 mM ATP,